Split-cycle and tape amplification

ABSTRACT

Methods and compositions are provided for improved nucleic acid amplification assays.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ProvisionalApplication No. 62/273,210, filed Dec. 30, 2015, the contents of whichare hereby incorporated by reference in the entirety for any and allpurposes.

BACKGROUND OF THE INVENTION

Quantitative detection of target sequences (e.g., alleles,polymorphisms, etc.) in a nucleic acid sample is useful in a variety ofcontexts. For example, detection of rare target sequences can be usefulfor early, benign, or malignant tumor detection or monitoring; prenataldiagnostics, such as non-invasive fetal diagnostics; detection of viralor bacterial infection; environmental monitoring, and the like. In somecases, such detection requires a high level of sensitivity, accuracy,and precision in order to detect low abundance target sequences in abackground of highly abundant non-target sequences.

BRIEF SUMMARY OF THE INVENTION

Described herein are methods and compositions for quantitative detectionof sequences in a nucleic acid sample. The methods typically involvepartitioning of a nucleic acid sample into a large number of mixturepartitions in discrete reaction chambers (e.g., wells, channels,droplets, etc.). In one aspect, the partitioned sample is analyzed within a split-cycle assay in which a pair of allele-specific amplificationprimers having a relatively low annealing temperature are used to appenda high-temperature primer binding site to the target sequence in aninitial set of nucleic acid amplification cycles. The initial set ofnucleic acid amplification cycles include a low temperature annealingstep. In subsequent nucleic acid amplification cycles, a pair offlanking amplification primers that hybridize to the high-temperatureprimer binding site provide high fidelity and highly specificamplification. The subsequent set of nucleic acid amplification cyclesinclude a higher temperature annealing step.

In another aspect, the partitioned sample is analyzed with in asplit-cycle assay in which a pair of allele-specific amplificationprimers having a relatively high annealing temperature are used toappend a low-temperature primer binding site to the target sequence inan initial set of nucleic acid amplification cycles. The initial set ofnucleic acid amplification cycles include a relatively high temperatureannealing step. In subsequent nucleic acid amplification cycles, a pairof flanking amplification primers that hybridize to the low-temperatureprimer binding site provide high fidelity and highly specificamplification. The subsequent set of nucleic acid amplification cyclesinclude a relatively lower temperature annealing step.

In another aspect, the partitioned sample is analyzed in a TaggedAmplicon Primer Extension (TAPE) reaction. In a TAPE reaction, targetsequences are amplified using a pair of 5′-tailed amplification primers,one 5′-tailed forward primer and one 5′-tailed reverse primer. The tailsof the 5′-tailed forward and reverse primers are reverse complements ofeach other. The amplification reaction therefore generates ampliconsthat incorporate the tags (i.e., tagged amplicons). The resulting taggedamplicons have 3′ ends that are reverse complements each other, andtherefore work as primers. Thus, the reaction self-generates primers,preventing primer depletion during the amplification reaction. In someembodiments, TAPE primers can be used in a split-cycle assay thatfurther includes flanking primers configured to hybridize to theamplicon tags.

In another aspect, the present invention provides a plurality of mixturepartitions, the individual mixture partitions comprising: i) amutation-specific 5′-tailed primer pair, wherein the mutation-specific5′-tailed primer pair hybridizes to and specifically amplifies targetDNA template molecules from a nucleic acid sample that comprise a mutanttarget sequence, if present, wherein the primers of the mutationspecific 5′-tailed primer pair comprise: a) a 3′ hybridization region ofat least 10 nucleotides in length and less than 30 nucleotides in lengththat specifically hybridizes to the mutant target sequence; and b) amutation-specific 5′ tail region of at least 10 nucleotides in lengthand no more than 30 nucleotides in length that does not hybridize to anynucleic acid fragments in the nucleic acid sample; ii) a wild-typespecific 5′-tailed primer pair, wherein the wild-type specific 5′-tailedprimer pair hybridizes to and specifically amplifies target DNA templatemolecules comprising a wild-type target sequence, if present, whereinthe primers of the wild-type specific 5′-tailed primer pair comprise: a)a 3′ hybridization region of at least 10 nucleotides in length and lessthan 30 nucleotides in length that specifically hybridizes to thewild-type target sequence; and b) a wild-type specific 5′ tail region ofat least 10 nucleotides in length and no more than 30 nucleotides thatdoes not hybridize to any nucleic acid fragments in the nucleic acidsample, wherein the wild-type specific 5′ tail region is a differentsequence than the mutation-specific 5′ tail region; and iii) a mutationspecific flanking primer pair, wherein the mutation specific flankingprimer pair hybridizes to and specifically amplifies ampliconscomprising the 5′ tail regions of the mutation specific 5′-tailed primerpair, if present; iv) a wild-type specific flanking primer pair, whereinthe wild-type specific flanking primer pair hybridizes to andspecifically amplifies amplicons comprising the 5′ tail regions of thewild-type specific 5′-tailed primer pair, if present; and v) athermostable polymerase, wherein at least about 1-10 of the mixturepartitions of the plurality of mixture partitions contains a target DNAtemplate molecule that comprises a wild-type or mutant target sequence;and at least about 3-10 of the mixture partitions of the plurality ofmixture partitions do not contain the target DNA template molecule.

In another aspect, the present invention provides a method forquantitating a frequency of wild-type and mutant target nucleic acidfragments in a nucleic acid sample, the method comprising: A) forming aplurality of mixture partitions of any one of the preceding claims; B)incubating the mixture partitions under thermal cycling conditionssuitable for amplification of the target DNA template molecules by apolymerase chain reaction, wherein the thermal cycling conditionscomprise a first set of temperature cycles and a second set oftemperature cycles, wherein the second set of temperature cyclescomprises an annealing temperature that is at least 5° C. higher orlower than an annealing temperature of the first set of temperaturecycles; C) detecting the presence or absence of amplified target DNAtemplate in the mixture partitions, wherein the detecting comprises,thereby determining: i) a number of wild-type mixture partitionscomprising amplified target DNA template consisting of wild-type targetsequence; ii) a number of mutant mixture partitions comprising amplifiedtarget DNA template consisting of mutant target sequence; and iii) anumber of double-positive mixture partitions comprising amplified targetDNA comprising mutant and wild-type target sequence; and D) determiningfrom the number of wild-type, mutant, and double-positive mixturepartitions the frequency of wild-type and mutant target nucleic acidfragments in the nucleic acid sample.

In another aspect, the present invention provides a reaction mixture forperforming a tagged amplicon primer extension (TAPE) nucleic acidamplification reaction, the mixture comprising: i) a target DNA templatemolecule from a nucleic acid sample, wherein the target DNA templatemolecule comprises a target sequence; ii) a forward primer comprising:a) a 3′ hybridization region of at least 10 nucleotides in length and nomore than 30 nucleotides in length that is configured to specificallyhybridize to the target sequence of the target DNA template molecule andgenerate a first primer extension product in the nucleic acidamplification reaction; and b) a 5′ tail region of at least 10nucleotides in length that is not complementary to the target sequenceof the target DNA template molecule; iii) a reverse primer comprising:a) a 3′ hybridization region of at least 10 nucleotides in length and nomore than 30 nucleotides in length that is configured to specificallyhybridize to the first primer extension product and generate a secondprimer extension product in in the nucleic acid amplification reaction;and b) a 5′ tail region of at least 10 nucleotides in length that is notcomplementary to the target sequence of the target DNA templatemolecule, wherein the 5′ tail region of the reverse primer is a reversecomplement of the 5′ tail region of the forward primer; and iv) athermostable polymerase.

In another aspect, the present invention provides a plurality of mixturepartitions, the individual mixture partitions comprising any one of theforegoing reaction mixtures.

In another aspect, the present invention provides a method forperforming a tagged amplicon primer extension (TAPE) nucleic acidamplification reaction, the method comprising: i) forming any one of theforegoing reaction mixtures, or any one of the foregoing pluralities ofreaction mixtures; ii) hybridizing a forward primer to a target sequenceof a target DNA template molecule; iii) extending the hybridized forwardprimer with a polymerase, thereby generating a first primer extensionproduct; iv) hybridizing a reverse primer to the first primer extensionproduct; v) extending the hybridized reverse primer with the polymerase,thereby generating a second primer extension product; vi) hybridizingthe forward primer to the second primer extension product; v) extendingthe forward primer hybridized to the second primer extension productwith the polymerase, thereby generating a third primer extensionproduct, wherein the second and third primer extension products form afirst double-stranded amplicon, wherein the first double-strandedamplicon comprises two complementary strands having 3′ and 5′ ends,wherein the 3′ ends are reverse complements of each other, and the 5′ends are reverse complements of each other.

In another aspect, the present invention provides a method forperforming a tagged amplicon primer extension (TAPE) nucleic acidamplification reaction, the method comprising: i) forming any one of theforegoing reaction mixtures; ii) hybridizing: a) a mutant-specificforward primer to a mutant target sequence of a target DNA templatemolecule, if present; and b) a wild-type specific forward primer to awild-type target sequence of a target DNA template molecule; iii)extending the hybridized forward primer(s) with a polymerase, therebygenerating a mutant first primer extension product if the mutant targetsequence is present and a wild-type first primer extension product; iv)hybridizing: a) a mutant-specific reverse primer to the mutant firstprimer extension product, if present; and b) a wild-type specificreverse primer to the wild-type first primer extension product; v)extending the hybridized reverse primer(s) with the polymerase, therebygenerating a mutant second primer extension product if the mutant targetsequence is present, and a wild-type second primer extension product;vi) hybridizing the forward primer(s) to the second primer extensionproduct(s), if present; v) extending the forward primer(s) hybridized tothe second primer extension product(s) with the polymerase, therebygenerating a mutant third primer extension product, if the mutant targetsequence is present and a wild-type third primer extension product,wherein: the second and third mutant primer extension products form amutant double-stranded amplicon, if the mutant target sequence ispresent, wherein the mutant double-stranded amplicon comprises twocomplementary strands having 3′ and 5′ ends, wherein the 3′ ends arereverse complements of each other, and the 5′ ends are reversecomplements of each other; and the second and third wild-type primerextension products form a wild-type double-stranded amplicon, whereinthe wild-type double-stranded amplicon comprises two complementarystrands having 3′ and 5′ ends, wherein the 3′ ends of the firstwild-type double-stranded amplicon are reverse complements of each otherand the 5′ ends of the first wild-type double-stranded amplicon arereverse complements of each other.

In another aspect, the present invention provides A method forquantitative rare mutation detection, the method comprising: i)providing any one of the foregoing pluralities of mixture partitions;ii) performing a split-cycle assay, TAPE assay, or combination thereofto separately detect wild-type and mutant target DNA template moleculesin the plurality of mixture partitions, thereby detecting a number ofmixture partitions that are positive for a presence of the mutant butnot the wild-type target DNA template, a number of mixture partitionsthat are positive for a presence of the wild-type but not the mutanttarget DNA template, a number of mixture partitions that are positivefor the presence of both the mutant and wild-type target DNA template,and a number of mixture partitions that are negative for the presence ofmutant and wild-type target DNA template; and iii) determining thefrequency of the mutant sequence in the nucleic acid sample from thenumber of single-positive, double-positive, and negative mixturepartitions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: illustrates exemplary primers and probes for performing anallele-specific split-cycle assay to simultaneously detect a mutant andwild-type target sequence. In this embodiment, the assay uses amutation-specific 5′-tailed forward primer (Mutation-specific F1),5′-tailed reverse primer (Mutation-specific R1), fluorescently labeledflanking forward primer (Mutation-specific flanking F1), flankingreverse primer (Mutation-specific flanking R1), and sink oligonucleotide(Mutant-specific sink), to amplify and detect mutant target sequence.The assay further uses a wild-type specific 5′-tailed forward primer(Wild-type specific F2), 5′-tailed reverse primer (Wild-type specificR2), fluorescently labeled flanking forward primer (Wild-type specificflanking F2), flanking reverse primer (Wild-type specific flanking R2),and sink oligonucleotide (Wild-type specific sink), to amplify anddetect wild-type target sequence. FAM and HEX refer to differentiallydetectable fluorescein derivatives. IABkFQ refers to an Iowa Blackfluorophore quencher. In this embodiment, the flanking primers have ahigher annealing temperature than the 5′-tailed primers, and thesplit-cycle assay would include a first set of temperature cycles havingan annealing temperature and a second set of temperature cycles havingan annealing temperature that is higher than the second set. In someembodiments, a different probe chemistry, such as an hydrolysis probe oran intercalating dye, can be used in addition or in the alternative tonucleic acid probes.

FIG. 2: illustrates exemplary primers and probes for performing anallele-specific split-cycle assay to simultaneously detect a mutant andwild-type target sequence. In this embodiment, the assay uses amutation-specific 5′-tailed forward primer (Mutation-specific F1),5′-tailed reverse primer (Mutation-specific R1), flanking forward primer(Mutation-specific flanking F1), flanking reverse primer(Mutation-specific flanking R1), and probe (Mutant-specific Probe), toamplify and detect mutant target sequence. The assay further uses awild-type specific 5′-tailed forward primer (Wild-type specific F2),5′-tailed reverse primer (Wild-type specific R2), flanking forwardprimer (Wild-type specific flanking F2), flanking reverse primer(Wild-type specific flanking R2), and probe (Wild-type specific Probe),to amplify and detect wild-type target sequence. FAM and HEX refer todifferentially detectable fluorescein derivatives. IABkFQ3′ refers to anIowa Black fluorophore quencher. In this embodiment, the flankingprimers have a lower annealing temperature than the 5′-tailed primers,and the split-cycle assay would include a first set of temperaturecycles having an annealing temperature and a second set of temperaturecycles having an annealing temperature that is lower than the secondset. In some embodiments, a different probe chemistry, such as afluorescently labeled flanking primer and short complementary quencheras in FIG. 1, or an intercalating dye, can be used in addition or in thealternative to nucleic acid probes.

FIGS. 3a-b : illustrates exemplary primers and amplification reactionsfor performing a TAPE assay. a) In this embodiment, the assay uses a5′-tailed forward primer (F1) and 5′-tailed reverse primer (R1) toamplify a target sequence. An hydrolysis probe (Wild-type Probe) labeledwith the HEX fluorophore and a blackhole quencher (BHQ) is used todetect amplification (e.g., step 3) and amplicons (e.g., step 4). Instep 1, primer F1 hybridizes to a target strand of the targetdouble-stranded DNA molecule, and a polymerase reaction extends theprimer. In step 2, primer R1 hybridizes to the extended F1 primer and apolymerase reaction extends the primer. In step 3, primer F1 hybridizesto the extended R1 primer and a polymerase reaction extends the primer,thereby generating a tagged double-stranded amplicon having 3′ ends thatare reverse complements of each other. Step 4 illustrates use of thestrands of the tagged amplicon as primers to generate an second taggedamplicon having 3′ ends that are reverse complements of each other. Thestrands of the second tagged amplicon can also function as primers, andfurther amplification cycles can generate further tagged ampliconshaving 3′ ends that are reverse complements of each other and functionas primers. In this example, 3′ to 5′ exonuclease activity in thereaction mixture degrades the 3′ ends of the target molecule, but 3′ to5′ exonuclease activity is not an essential element of all TAPEreactions described herein.

b) In this embodiment, the assay uses a 5′-tailed forward primer (F1)and 5′-tailed reverse primer (R1) to amplify a target sequence. One5′-tailed primer is labeled with a fluorophore (FAM). The assay furtherincludes a black hole quencher (BHQ)-labeled sink oligonucleotide (Sink)that is complementary to the 5′ end of the labeled primer to detectamplification. In step 1, primer F1 hybridizes to a target strand of thetarget double-stranded DNA molecule, and a polymerase reaction extendsthe primer. In step 2, primer R1 hybridizes to the extended F1 primerand a polymerase reaction extends the primer. In step 3, primer F1hybridizes to the extended R1 primer and a polymerase reaction extendsthe primer, thereby generating a tagged double-stranded amplicon having3′ ends that are reverse complements of each other. Step 4 illustratesuse of the strands of the tagged amplicon as primers to generate ansecond tagged amplicon having 3′ ends that are reverse complements ofeach other. The strands of the second tagged amplicon can also functionas primers, and further amplification cycles can generate further taggedamplicons having 3′ ends that are reverse complements of each other andfunction as primers. In this example, 3′ to 5′ exonuclease activity inthe reaction mixture degrades the 3′ ends of the target molecule, but 3′to 5′ exonuclease activity is not an essential element of all TAPEreactions described herein.

FIG. 4: illustrates a comparison between typical results of a raremutation detection assay using conventional droplet digitalamplification methods (Left) and a split-cycle, TAPE, or split-cycle andTAPE assay (Right). Channel 1 represents a detection signal from amutant-specific hydrolysis probe. Channel 2 represents the signal from awild-type specific hydrolysis probe. In the conventional assay, primerdepletion and competition result in double-positive droplets that smearinto the wild-type and/or mutant only (single-positive) detectionregions, decreasing the accuracy of the assay. In comparison, methodsdescribed herein result well-separated single- and double-positiveregions.

FIG. 5: illustrates exemplified data from a split-cycle droplet digitalPCR assay performed using methods described herein for rare mutationdetection. (Left) As a control, PIK3CA wild-type and mutant E542K assaysusing a standard PCR amplification protocol with Taqman detection todifferentially detect the single nucleotide polymorphism were performed.Channel 1 represents a detection signal from a mutant-specifichydrolysis probe. Channel 2 represents the signal from a wild-typespecific hydrolysis probe. In the conventional assay, primer depletionand competition resulted in double-positive droplets that smeared intothe wild-type and/or mutant only (single-positive) detection regions,decreasing the accuracy of the assay. In comparison, split-cycle methodsdescribed herein resulted in well-separated single- and double-positiveregions.

DEFINITIONS

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art. See, e.g., Lackie, DICTIONARY OF CELL AND MOLECULARBIOLOGY, Elsevier (4^(th) ed. 2007); Sambrook et al., MOLECULAR CLONING,A LABORATORY MANUAL, Cold Spring Harbor Lab Press (Cold Spring Harbor,N.Y. 1989). The term “a” or “an” is intended to mean “one or more.” Theterm “comprise,” and variations thereof such as “comprises” and“comprising,” when preceding the recitation of a step or an element, areintended to mean that the addition of further steps or elements isoptional and not excluded. Any methods, devices and materials similar orequivalent to those described herein can be used in the practice of thisinvention. The following definitions are provided to facilitateunderstanding of certain terms used frequently herein and are not meantto limit the scope of the present disclosure.

As used herein, the term “complement” or “complementary” in reference toa primer, oligonucleotide, amplicon, or target sequence, or region ofthe primer, oligonucleotide, amplicon, or target sequence, can includethe reverse complement or reverse complementarity as required tomaintain functionality of the primer, oligonucleotide, amplicon, ortarget sequence or region thereof.

The term “amplification reaction” refers to any in vitro means formultiplying the copies of a target sequence of nucleic acid in a linearor exponential manner. Such methods include but are not limited topolymerase chain reaction (PCR); DNA ligase chain reaction (see U.S.Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods andApplications (Innis et al., eds, 1990)) (LCR); QBeta RNA replicase andRNA transcription-based amplification reactions (e.g., amplificationthat involves T7, T3, or SP6 primed RNA polymerization), such as thetranscription amplification system (TAS), nucleic acid sequence basedamplification (NASBA), and self-sustained sequence replication (3 SR);isothermal amplification reactions (e.g., single-primer isothermalamplification (SPIA)); as well as others known to those of skill in theart.

“Amplifying” refers to a step of submitting a solution to conditionssufficient to allow for amplification of a polynucleotide if all of thecomponents of the reaction are intact. Components of an amplificationreaction include, e.g., primers, a polynucleotide template, polymerase,nucleotides, and the like. The term “amplifying” typically refers to an“exponential” increase in target nucleic acid. However, “amplifying” asused herein can also refer to linear increases in the numbers of aselect target sequence of nucleic acid, such as is obtained with cyclesequencing or linear amplification.

The term “amplification reaction mixture” refers to an aqueous solutioncomprising the various reagents used to amplify a target nucleic acid.These include enzymes, aqueous buffers, salts, amplification primers,target nucleic acid, and nucleoside triphosphates. Amplificationreaction mixtures may also further include stabilizers and otheradditives to optimize efficiency and specificity. Depending upon thecontext, the mixture can be either a complete or incompleteamplification reaction mixture

“Polymerase chain reaction” or “PCR” refers to a method whereby aspecific segment or subsequence of a target double-stranded DNA, isamplified in a geometric progression. PCR is well known to those ofskill in the art; see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202; andPCR Protocols: A Guide to Methods and Applications, Innis et al., eds,1990. Exemplary PCR reaction conditions typically comprise either two orthree step cycles. Two step cycles have a denaturation step followed bya hybridization/elongation step. Three step cycles comprise adenaturation step followed by a hybridization step followed by aseparate elongation step.

A “primer” refers to a polynucleotide sequence that hybridizes to asequence on a target nucleic acid and serves as a point of initiation ofnucleic acid synthesis. Primers can be of a variety of lengths and areoften less than 50 nucleotides in length, for example 12-30 nucleotides,in length. The length and sequences of primers for use in PCR can bedesigned based on principles known to those of skill in the art, see,e.g., Innis et al., supra. Primers can be DNA, RNA, or a chimera of DNAand RNA portions. In some cases, primers can include one or moremodified or non-natural nucleotide bases. In some cases, primers arelabeled. A “pair” of primers, “primer pair,” “pair of forward andreverse primers,” and the like refers to a first and second primer,wherein one primer is a “forward” primer and the second primer is a“reverse” primer configured to hybridize to a target sequence andamplify the target sequence under PCR amplification conditions.

A nucleic acid, or a portion thereof, “hybridizes” to another nucleicacid under conditions such that non-specific hybridization is minimal ata defined temperature in a physiological buffer (e.g., pH 6-9, 25-150 mMchloride salt). In some cases, a nucleic acid, or portion thereof,hybridizes to a conserved sequence shared among a group of targetnucleic acids. In some cases, a primer, or portion thereof, canhybridize to a primer binding site if there are at least about 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 contiguous complementarynucleotides, including “universal” nucleotides that are complementary tomore than one nucleotide partner. Alternatively, a primer, or portionthereof, can hybridize to a primer binding site if there are fewer than1 or 2 complementarity mismatches over at least about 12, 13, 14, 15,16, 17, or 18 contiguous nucleotides. In some embodiments, the definedtemperature at which specific hybridization occurs is room temperature.In some embodiments, the defined temperature at which specifichybridization occurs is higher than room temperature. In someembodiments, the defined temperature at which specific hybridizationoccurs is at least about 37, 40, 42, 45, 50, 55, 60, 65, 70, or 75° C.In some embodiments, the defined temperature at which specifichybridization occurs is 37, 40, 42, 45, 50, 55, 60, 65, 70, or 75° C.

A “template” refers to a polynucleotide sequence that comprises thepolynucleotide to be amplified, flanked by a pair of primerhybridization sites or adjacent to a primer hybridization site. Thus, a“target template” comprises the target polynucleotide sequence adjacentto at least one hybridization site for a primer. A “target DNA template”comprises a target DNA polynucleotide sequence. In some cases, a “targettemplate” comprises the target polynucleotide sequence flanked by ahybridization site for a “forward” primer and a “reverse” primer. Atarget template molecule, such as a target DNA template molecule, cancomprise a mutant sequence or a wild-type sequence. A plurality oftarget DNA template molecules can comprise a plurality of wild-typesequences, a plurality of mutant sequences, or a combination thereof.

As used herein, “nucleic acid” means DNA, RNA, single-stranded,double-stranded, or more highly aggregated hybridization motifs, and anychemical modifications thereof. Modifications include, but are notlimited to, those providing chemical groups that incorporate additionalcharge, polarizability, hydrogen bonding, electrostatic interaction,points of attachment and functionality to the nucleic acid ligand basesor to the nucleic acid ligand as a whole. Such modifications include,but are not limited to, peptide nucleic acids (PNAs), phosphodiestergroup modifications (e.g., phosphorothioates, methylphosphonates),2′-position sugar modifications, 5-position pyrimidine modifications,8-position purine modifications, modifications at exocyclic amines,substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil;backbone modifications, methylations, unusual base-pairing combinationssuch as the isobases, isocytidine and isoguanidine and the like. Nucleicacids can also include non-natural bases, such as, for example,nitroindole. Modifications can also include 3′ and 5′ modificationsincluding but not limited to capping with a fluorophore (e.g., quantumdot or fluorescent organic dye) or another moiety.

“Probe” refers to an oligonucleotide which binds through complementarybase pairing to a subsequence of a target nucleic acid. It will beunderstood by one of skill in the art that probes will typicallysubstantially bind target sequences lacking complete complementaritywith the probe sequence depending upon the stringency of thehybridization conditions. The probes are typically directly labeled(e.g., with isotopes or fluorescent moieties) or indirectly labeled suchas with digoxigenin or biotin. In some cases, the probe is a detectablylabeled primer. By assaying for the presence or absence of the probe,and/or its detectable label, one can detect the presence or absence ofthe target. In some cases, the probe binds target sequences havingcomplete complementary with the probe, but does not bind targets thatdiffer from the target sequence by a single nucleotide or more.

“Sink oligonucleotide” refers to a short oligonucleotide that iscomplementary to a probe or primer that is detectably labeled with aphotoluminophore. The sink oligonucleotide is labeled with an energytransfer partner that quenches the detectable signal of thecomplementary detectably labeled probe or primer when hybridizedthereto. Detection of a presence or absence of an unquenched signal fromthe photoluminophore indicates the presence or absence respectively of atarget nucleic acid. The quenching can be reduced or eliminated bydegradation of the sink oligonucleotide or otherwise disrupting thehybridization complex between the sink oligonucleotide and detectablylabeled primer.

As used herein, the term “partitioning” or “partitioned” refers toseparating a sample into a plurality of portions, or “partitions.”Partitions can be solid or fluid. In some embodiments, a partition is asolid partition, e.g., a micro channel. In some embodiments, a partitionis a fluid partition, e.g., a droplet. In some embodiments, a fluidpartition (e.g., a droplet) is a mixture of immiscible fluids (e.g.,water and oil), or an emulsion. In some embodiments, a fluid partition(e.g., a droplet) is an aqueous droplet that is surrounded by animmiscible carrier fluid (e.g., oil). In other embodiments, a fluidpartition is an aqueous droplet that is physically or chemicallyseparated from adjacent aqueous droplets such that the contents of onedroplet does not diffuse into adjacent droplets.

A “polymerase” refers to an enzyme that performs template-directedsynthesis of polynucleotides, e.g., DNA and/or RNA. The term encompassesboth the full length polypeptide and a domain that has polymeraseactivity. DNA polymerases are well-known to those skilled in the art,including but not limited to DNA polymerases isolated or derived fromPyrococcus furiosus, Thermococcus litoralis, and Thermotoga maritime, ormodified versions thereof. Additional examples of commercially availablepolymerase enzymes include, but are not limited to: Klenow fragment (NewEngland Biolabs® Inc.), Taq DNA polymerase (QIAGEN), 9° N™ DNApolymerase (New England Biolabs® Inc.), Deep Vent™ DNA polymerase (NewEngland Biolabs® Inc.), Manta DNA polymerase (Enzymatics®), Bst DNApolymerase (New England Biolabs® Inc.), and phi29 DNA polymerase (NewEngland Biolabs® Inc.).

Polymerases include both DNA-dependent polymerases and RNA-dependentpolymerases such as reverse transcriptase. At least five families ofDNA-dependent DNA polymerases are known, although most fall intofamilies A, B and C. Other types of DNA polymerases include phagepolymerases. Similarly, RNA polymerases typically include eukaryotic RNApolymerases I, II, and III, and bacterial RNA polymerases as well asphage and viral polymerases. RNA polymerases can be DNA-dependent andRNA-dependent.

The terms “label,” “detectable label, “detectable moiety,” and liketerms refer to a composition detectable by spectroscopic, photochemical,biochemical, immunochemical, chemical, or other physical means. Forexample, useful labels include fluorescent dyes (fluorophores),luminescent agents, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, ³²P and other isotopes, haptens,and proteins which can be made detectable, e.g., by incorporating aradiolabel into the peptide or used to detect antibodies specificallyreactive with the peptide. The term includes combinations of singlelabeling agents, e.g., a combination of fluorophores that provides aunique detectable signature, e.g., at a particular wavelength orcombination of wavelengths. Any method known in the art for conjugatinglabel to a desired agent may be employed, e.g., using methods describedin Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., SanDiego.

DETAILED DESCRIPTION OF THE INVENTION I. Overview

Described herein are methods and compositions for performing split-cycleand tagged amplicon primer extension (TAPE) amplification of nucleicacids from a sample. The split-cycle and TAPE methods are not mutuallyexclusive. Accordingly, described herein are methods and composition forperforming split-cycle and TAPE amplification in an nucleic acidamplification reaction or a plurality of nucleic acid amplificationreactions.

The methods and compositions can, e.g., reduce competition for primersbetween mutant and wild-type target templates in a nucleic acidamplification reaction. The methods and compositions can additionally oralternatively reduce or eliminate primer depletion in a nucleic acidamplification reaction by, e.g., generating additional primers in situ.In some cases, the methods and compositions described herein can lowerthe error-rate of quantitative nucleic acid amplification by loweringthe number of mutations generated by the polymerase in an amplificationreaction.

In some cases, the methods and compositions described herein involvequantitative analysis of multiple different target templates (e.g.,two-dimensional analysis of mutant and wild-type target sequence) in apartitioned nucleic acid sample. In such cases, the methods andcompositions described herein can, e.g., provide increased separationbetween single-positive and double-positive partitions, thus allowingeasier thresholding and lowering the error rate of the analysis.

II. Compositions

Described herein are compositions for performing nucleic acidamplification reactions.

a. Split-Cycle Compositions

In one aspect, the composition is a primer or set of primers forperforming a split-cycle amplification reaction. Primers for performingsplit-cycle amplification reactions include, but are not limited to,5′-tailed primers and flanking primers. 5′-tailed primers are typicallyused in pairs of forward and reverse 5′-tailed primers to amplify anintervening target sequence of a target template. The 5′-tailed primersinclude a 3′ hybridizing region that specifically hybridizes to thetarget sequence and functions as a primer in a polymerase-mediatedprimer extension reaction. The 3′ hybridizing regions of the forward andreverse 5′-tailed primers are typically configured to hybridize toopposite strands of a target template such that the 3′ ends are orientedtoward each other. As such, the primers can be used to amplify thetarget template and produce a pair of reverse complementary primerextension products, i.e., amplicons.

The primer extension products and amplicons of such an amplificationreaction include the target sequence and 5′ tail sequences of the5′-tailed primers. The 5′-tail regions of the 5′-tailed primers,corresponding primer extension products, and amplicons resulting fromamplification with the 5′-tailed primers can be selected to serve asprimer binding sites for flanking primers. In some cases, the 5′-tailregions are selected to comprise a pair of unique, or substantiallyunique, sequences as compared to the oligonucleotide sequences of theanalyzed nucleic acid sample. For example, if the nucleic acid sample isa sample of nucleic acid from a human subject, the 5′-tail regions canbe selected to be unique as compared to the sequence of the humangenome.

In some cases, the 5′-tail regions of the 5′-tailed primers of a5′-tailed primer pair (i.e., a forward 5′-tailed primer andcorresponding reverse 5′-tailed primer) can be reverse complements ofeach other. In such cases, the 5′-tailed primers can support taggedamplicon primer extension (TAPE). In a TAPE reaction, the 5′-tailedprimers generate amplicons that have 3′ ends that serve as primers insubsequent amplification cycles. Compositions and methods for TAPE arefurther described herein.

Flanking primers are typically used in pairs of forward and reverseflanking primers to hybridize to the 5′-tail regions of the primerextension products and amplicons produced by amplification of the targettemplates with the forward and reverse 5′-tailed primer pairs. Theflanking primers are configured to hybridize to opposite strands of the5′-tailed primer extension products target template such that the 3′ends are oriented toward each other. Thus, the split-cycle amplificationincludes a first set of amplification cycles in which 5′-tailed forwardand reverse primers hybridize to target templates and are extended toproduce 5′-tailed primer extension products and amplicons, and a secondset of amplification cycles in which flanking primers hybridize to the5′-tailed primer extension products and are extended to produceamplicons.

i. 5′-Tailed Primers for Split-Cycle Up Amplification

In some embodiments, the 5′-tailed forward and 5′-tailed reverse primersanneal to the target DNA template at a relatively low temperature ascompared to the annealing of the flanking primers to the products of thefirst set of amplification cycles. Split-cycle amplification in whichthe annealing temperature of the 5′-tailed primers is lower than theannealing temperature of the flanking primers is referred to assplit-cycle up amplification because the annealing temperature is raisedup in a second set of amplification cycles. In some cases, thisdifference in annealing temperatures can provide for control of whetherextension of 5′-tailed primers or extension of flanking primers ispredominantly performed in a reaction in a given cycle. For example, asexplained in further detail below, 5′-tailed primer extension can befavored during a first set of amplification cycles having relatively lowtemperature annealing, and optionally primer extension, stages.Similarly, flanking primer extension can be favored during a second setof amplification cycles having relatively high temperature annealing,and optionally primer extension stages.

Thus, the hybridization regions of the 5′-tailed primers can be shorterthan the hybridization regions of the flanking primers. In someembodiments, the hybridization regions of the 5′-tailed primers are atleast about 8 nucleotides in length. In some cases, the hybridizationregions of the 5′-tailed primers are 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 nucleotides in length. Insome cases, the hybridization regions of the 5′-tailed primers are fromabout 8 to about 45, from about 9 to about 40, from about 8 to about 35,from about 8 to about 30, from about 9 to about 30, from about 10 toabout 30, from about 11 to about 25, from about 12 to about 25, or fromabout 15 to about 25 nucleotides in length. In some cases, thehybridization regions of the 5′-tailed primers are from about 8 to about15, from about 9 to about 15, from about 10 to about 15, from about 12to about 15, from about 8 to about 20, from about 9 to about 20, fromabout 10 to about 20, from about 12 to about 20, or from about 15 toabout 20 nucleotides in length. In some cases, the hybridization regionsof the 5′-tailed primers are from about 8 to about 25, from about 9 toabout 25, from about 10 to about 25, from about 12 to about 25, fromabout 15 to about 25, from about 18 to about 25, from about 8 to about30, from about 9 to about 30, from about 10 to about 30, from about 12to about 30, from about 15 to about 30, from about 18 to about 30, orfrom about 20 to about 30 nucleotides in length.

Additionally, or alternatively, the hybridization regions of the5′-tailed primers can have a lower G-C content, or be modified toexhibit a reduce annealing temperature as compared to the flankingprimers in the split-cycle reaction. In some cases, the hybridizationregions of the 5′-tailed primers have an annealing temperature that isat least 1° C. lower than the lowest hybridization temperature of theflanking primers in the split-cycle reaction. In some cases, thehybridization regions of the 5′-tailed primers have an annealingtemperature that is, is about, is at least, or is at least about, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, or 45° C. lower than the lowest hybridizationtemperature of the flanking primers in the split-cycle reaction. In somecases, the hybridization regions of the 5′-tailed primers have anannealing temperature that is from about 1° C. to about 45° C., fromabout 2° C. to about 35° C., from about 3° C. to about 30° C., fromabout 5° C. to about 20° C., from about 5° C. to about 15° C., or fromabout 5° C. to about 10° C. less than the lowest hybridizationtemperature of the flanking primers in the split-cycle reaction.

In some embodiments, the hybridization regions of the 5′-tailed primershave an annealing temperature of less than about 75° C. In someembodiments, the hybridization regions of the 5′-tailed primers have anannealing temperature of from about 30° C. to about 75° C., from about35° C. to about 70° C.; from about 40° C. to about 65° C.; from about45° C. to about 60° C.; or from about 45° C. to about 55° C. In someembodiments, the hybridization regions of the 5′-tailed primers have anannealing temperature of, or at least, of about, or of at least about,35, 40, 45, 50, 55, 60, or 65° C. (e.g., and less than about 75° C.).

The 5′-tail regions of the 5′-tailed primers can be configured toprovide a primer binding site for the corresponding flanking primer thathas a higher annealing temperature than the annealing temperature of the5-tailed primers to the target DNA template. Additionally oralternatively, the 5′-tail regions can have a longer length innucleotides than the 3′ hybridization regions. In some embodiments, the5′-tail regions of the 5′-tailed primers are at least about 10nucleotides in length. In some cases, the 5′-tail regions of the5′-tailed primers are, are about, are at least, or are at least about,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or45 nucleotides in length. In some cases, the 5′-tail regions of the5′-tailed primers are from about 10 to about 45, from about 11 to about40, from about 12 to about 35, from about 10 to about 30, from about 11to about 30, from about 12 to about 30, or from about 15 to about 30nucleotides in length. In some cases, the 5′-tail regions of the5′-tailed primers are from about 12 to about 25, from about 15 to about25, from about 20 to about 25, from about 12 to about 30, from about 15to about 30, from about 20 to about 30, or from about 25 to about 30nucleotides in length. Additionally, or alternatively, the 5′-tailregions of the 5′-tailed primers can have a higher G-C content ascompared to the hybridization region. For example, the 5′-tail regioncan have, or have at least, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, or 15 more G or C nucleotides as compared to the hybridizationregion. As another example, the 5′-tail region can have from about 1 toabout 3, from about 1 to about 5, from about 1 to about 10, from about 2to about 3, from about 2 to about 5, or from about 2 to about 10 more Gor C nucleotides as compared to the hybridization region.

ii. 5′-Tailed Primers for Split-Cycle Down Amplification

In some embodiments, the 5′-tailed forward and 5′-tailed reverse primersanneal to the target DNA template at a relatively high temperature ascompared to the annealing of the flanking primers to the products of thefirst set of amplification cycles. Split-cycle amplification in whichthe annealing temperature of the 5′-tailed primers is higher than theannealing temperature of the flanking primers is referred to assplit-cycle down amplification because the annealing temperature islowered down in a second set of amplification cycles. In some cases,this difference in annealing temperatures can control whether extensionof 5′-tailed primers or extension of flanking primers is predominantlyperformed in a reaction in a given cycle. For example, as explained infurther detail below, 5′-tailed primer extension can be favored during afirst set of amplification cycles having relatively high temperatureannealing, and optionally primer extension, stages. Similarly, flankingprimer extension can be favored during a second set of amplificationcycles in which 5′-tailed primers are depleted and relatively lowtemperature annealing, and optionally primer extension stages, areemployed.

Thus, the hybridization regions of the 5′-tailed primers can be longerthan the hybridization regions of the flanking primers. In someembodiments, the hybridization regions of the 5′-tailed primers are atleast about 10 nucleotides in length. In some cases, the hybridizationregions of the 5′-tailed primers are, are about, are at least, or are atleast about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, or 45 nucleotides in length. In some cases, the hybridizationregions of the 5′-tailed primers are from about 12 to about 45, fromabout 15 to about 40, from about 18 to about 35, from about 20 to about30, from about 25 to about 30, from about 15 to about 30, from about 18to about 25, from about 20 to about 25, or from about 15 to about 25nucleotides in length.

Additionally, or alternatively, the hybridization regions of the5′-tailed primers can have a higher G-C content, or be modified toexhibit an increased annealing temperature as compared to the flankingprimers in the split-cycle reaction. In some cases, the hybridizationregions of the 5′-tailed primers have an annealing temperature that isat least 1° C. higher than the highest hybridization temperature of theflanking primers in the split-cycle reaction. In some cases, thehybridization regions of the 5′-tailed primers have an annealingtemperature that is, is about, is at least, or is at least about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, or 45° C. higher than the highest hybridizationtemperature of the flanking primers in the split-cycle reaction. In somecases, the hybridization regions of the 5′-tailed primers have anannealing temperature that is from about 1° C. to about 45° C., fromabout 2° C. to about 35° C., from about 3° C. to about 30° C., fromabout 5° C. to about 20° C., from about 5° C. to about 15° C., or fromabout 5° C. to about 10° C. higher than the highest hybridizationtemperature of the flanking primers in the split-cycle reaction. In somecases, the hybridization regions of the 5′-tailed primers have anannealing temperature that is from about 1° C. to about 5° C., fromabout 2° C. to about 5° C., from about 3° C. to about 5° C., from about1° C. to about 10° C., from about 2° C. to about 10° C., from about 3°C. to about 10° C., or from about 5° C. to about 10° C. higher than thehighest hybridization temperature of the flanking primers in thesplit-cycle reaction. In some cases, the hybridization regions of the5′-tailed primers have an annealing temperature that is from about 10°C. to about 25° C., from about 10° C. to about 30° C., from about 10° C.to about 35° C., from about 15° C. to about 25° C., from about 15° C. toabout 30° C., from about 15° C. to about 35° C., or from about 20° C. toabout 30° C. higher than the highest hybridization temperature of theflanking primers in the split-cycle reaction.

In some embodiments, the hybridization regions of the 5′-tailed primershave an annealing temperature of less than about 75° C. In someembodiments, the hybridization regions of the 5′-tailed primers have anannealing temperature of greater than about 50° C. (e.g., greater thanabout 50° C. and less than about 75° C.). In some embodiments, thehybridization regions of the 5′-tailed primers have an annealingtemperature of from about 45° C. to about 75° C., from about 50° C. toabout 70° C.; from about 55° C. to about 68° C.; from about 55° C. toabout 65° C.; or from about 55° C. to about 60° C. In some embodiments,the hybridization regions of the 5′-tailed primers have an annealingtemperature of, or of about, 50, 55, 60, 65, 68, 70, 72, or 75° C.

The 5′-tail regions of the 5′-tailed primers can be configured toprovide a primer binding site for the corresponding flanking primer thathas a lower annealing temperature than the annealing temperature of the5-tailed primers. Additionally or alternatively, the 5′-tail regions canhave a shorter length in nucleotides than the 3′ hybridization regions.In some embodiments, the 5′-tail regions of the 5′-tailed primers are atleast about 7 nucleotides in length. In some cases, the 5′-tail regionsof the 5′-tailed primers are, are about, or are at least about 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or45 nucleotides in length. In some cases, the 5′-tail regions of the5′-tailed primers are from about 7 to about 35, about 8 to about 30,about 9 to about 30, about 7 to about 30, about 8 to about 25, about 9to about 20, about 7 to about 20, about 8 to about 18, about 9 to about15 or about 8 to about 15, nucleotides in length. Additionally, oralternatively, the 5′-tail regions of the 5′-tailed primers can have alower G-C content as compared to the hybridization region. For example,the 5′-tail region can have, or have at least, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, or 15 fewer G or C nucleotides as compared to thehybridization region.

In some embodiments, the flanking primers have an annealing temperatureof at least about 45° C. In some embodiments, the flanking primers havean annealing temperature of from about 45° C. to about 80° C., fromabout 45° C. to about 75° C.; from about 45° C. to about 65° C.; fromabout 45° C. to about 60° C.; or from about 45° C. to about 55° C. Insome embodiments, the flanking primers have an annealing temperature of,or of about, 45, 50, 55, 60, 65, 70, or 75° C.

Flanking primers are typically the same length as the 5′-tail regions ofthe corresponding 5′-tailed primers. However, flanking primers can beshorter than the 5′-tail region or can contain additional nucleotides,detectable labels, etc. In some embodiments, the flanking primers are atleast about 8 nucleotides in length. In some cases, the flanking primersare 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, or 45 nucleotides in length. In some cases, the flanking primersare from about 7 to about 15, about 8 to about 15, about 9 to about 25,about 10 to about 45, about 11 to about 40, about 12 to about 35, about10 to about 30, about 11 to about 30, about 12 to about 30, or about 15to about 30, nucleotides in length.

Flanking primers can be modified at one or more positions to increase ordecrease annealing temperatures. Suitable modifications to increaseannealing temperature include, but are not limited to, one or more ofthe modifications selected from 2′ fluoro nucleosides, LNAs (lockednucleic acids), ZNAs (zip nucleic acids), and PNAs (Peptide NucleicAcids). Suitable modifications to decrease annealing temperatureinclude, but are not limited to, base and backbone modifications. Basemodifications that alter the Watson-Crick interaction between oppositebases include inosine, xanthosine, oxo-guanine, etc. Backbonemodifications such as acyclic nucleoside analogues lower the meltingtemperature without affecting the Watson-Crick interaction (Nielsen, P.,Dreiøe, L. H. & Wengel, J. Synthesis and evaluation ofoligodeoxynucleotides containing acyclic)). Another backbonemodification known to decrease annealing temperature can be consideredan abasic site (Hianik, T. et al. DNA-duplexes containing abasic sites:correlation between thermostability and acoustic wave properties.Analyst 131, 1161-1166 (2006)) or linker that completely lacks a base toform a pair with the opposite strand.

In some embodiments, one or more flanking primers can include adetectable label (e.g., fluorescent label). In some cases, split-cyclereaction mixtures in which one or more flanking primers include adetectable fluorescent label also include a short oligonucleotide thatincludes a quencher and is reverse complementary to the fluorescentlylabeled flanking primer. In some cases, such reaction mixtures include athermostable polymerase with strand-displacing activity. In some cases,amplification in the presence of a fluorescently labeled flankingprimer, quencher, and strand-displacing polymerase is performed with anAmplicon Mediated Probe assay (AMP assay). Such assays are describedfurther in US 2015/0,148,250, herein incorporated by reference in theentirety for all purposes. In some embodiments, flanking primers havinga detectable label conjugated thereto are useful for split-cycle downamplification reactions, in which the annealing temperature is loweredin the second set of amplification cycles. In some embodiments, flankingprimers having a detectable label conjugated thereto are useful forsplit-cycle up amplification reactions in which the annealingtemperature is raised in the second set of amplification cycles.

In some embodiments, 5′-tailed primers are provided in the split-cycleamplification reaction at a lower concentration as compared to flankingprimers. Lower concentration 5′-tailed primers can be useful, e.g., torapidly deplete 5′-tailed primers during early rounds of amplificationand ensure that later amplification cycles are predominantly performedby hybridization and extension of flanking primers. Thus, in someembodiments, the 5′-tailed primers are at a concentration that is lessthan about 50% (i.e., less than about ½) of the concentration of theflanking primers. As used herein, the term 50%, half, one-half, or ½, inreference to the concentration of a first pair off primers (e.g.,5′-tailed primers) to a second pair of primers (e.g., flanking primers)refers to a numerical concentration value for the first pair of primersthat is one-half the concentration value for the second pair of primers.Thus, for the purposes of illustration only, for a first pair of primersat 1 μM in a reaction mixture and at ½ the concentration of a secondpair of primers in the reaction mixture, the second pair of primers willbe at 2 μM. One of skill in the art will appreciate that an alternativepercentage or range of percentage of reduced or increased concentrationcan be similarly calculated and understood by those of ordinary skill inthe art. Generally, the individual primers of a primer pair are at, orat about, equimolar concentration with respect to each other.

In some embodiments, the 5′-tailed primers are at a concentration thatis from at least about 0.0001% to at least about ½ (i.e., 50%) of theconcentration of the flanking primers. In some embodiments, the5′-tailed primers are at a concentration that is from at least about0.001% to at least about ½ (i.e., 50%) of the concentration of theflanking primers. In some embodiments, the 5′-tailed primers are at aconcentration that is from at least about 0.01% to no more than about ½(i.e., 50%) of the concentration of the flanking primers. In someembodiments, the 5′-tailed primers are at a concentration that is fromat least about 0.1% to no more than about ½ (i.e., 50%) of theconcentration of the flanking primers. In some embodiments, the5′-tailed primers are at a concentration that is from at least about 1%to no more than about ½ (i.e., 50%) of the concentration of the flankingprimers. In some embodiments, the 5′-tailed primers are at aconcentration that is from at least about 5% to no more than about ½(i.e., 50%) of the concentration of the flanking primers. In someembodiments, the 5′-tailed primers are at a concentration that is fromat least about 10% to no more than about ½ (i.e., 50%) of theconcentration of the flanking primers. In some embodiments, the5′-tailed primers are at a concentration that is from at least about 15%to no more than about ½ (i.e., 50%) of the concentration of the flankingprimers. In some embodiments, the 5′-tailed primers are at aconcentration that is from at least about 20% to no more than about ½(i.e., 50%) of the concentration of the flanking primers. In some cases,the 5′-tailed primers are at a concentration that is, or is about, theconcentration of the flanking primers.

In some cases, relative primer concentrations, e.g., in combination withrelative annealing temperatures, of the 5′-tailed primers as compared tothe corresponding flanking primers can provide control of whetherextension of 5′-tailed primers or extension of flanking primers ispredominantly performed in a reaction in a given cycle. For example,low-temperature annealing 5′-tailed primers can be provided in asplit-cycle amplification reaction at a higher concentration thanhigh-temperature annealing flanking primers. Thus, amplification cycleshaving a low temperature annealing stage can predominantly extendhybridized 5′-tailed primers even though flanking primers can anneal atsuch temperatures, while high temperature annealing cycles canpredominantly extend hybridized flanking primers because annealing of5′-tailed primers at the higher annealing temperature is disfavored.

Thus, in some embodiments, the 5′-tailed primers are at a concentrationthat is from at least about 2-fold to at least about 10,000-fold higherthan the concentration of the flanking primers. In some embodiments, the5′-tailed primers are at a concentration that is from at least about2-fold and no more than about 10,000-fold higher than the concentrationof the flanking primers. In some cases, the 5′-tailed primers are at aconcentration that is, or is about, 5-fold; 10-fold; 15-fold; 20-fold,30-fold, 50-fold, 75-fold; 100-fold, 250-fold; 500-fold; or 1,000-foldhigher than the concentration of the flanking primers. In some cases,the 5′-tailed primers are at a concentration that is from at least about2-fold to no more than about 1,000-fold, from at least about 2-fold tono more than about 500-fold, from at least about 2-fold to no more thanabout 100-fold, from at least about 2-fold to no more than about50-fold, from at least about 2-fold to no more than about 10-fold, orfrom at least about 2-fold to no more than 5-fold higher than theconcentration of the flanking primers.

Similarly, high-temperature annealing 5′-tailed primers can be providedin a split-cycle amplification reaction at a lower concentration thanlow-temperature annealing flanking primers. Thus, initial amplificationcycles having a high temperature annealing stage can predominantlyextend and deplete hybridized 5′-tailed primers, while subsequent lowtemperature annealing cycles can predominantly extend hybridizedflanking primers because the tailed primers are depleted.

Thus, in some embodiments, the flanking primers are at a concentrationthat is from at least about 2-fold to at least about 10,000-fold higherthan the concentration of the 5′-tailed primers. In some embodiments,the flanking primers are at a concentration that is from at least about2-fold and no more than about 10,000-fold higher than the concentrationof the 5′-tailed primers. In some cases, the flanking primers are at aconcentration that is, or is about, 5-fold; 10-fold; 15-fold; 20-fold,30-fold, 50-fold, 75-fold; 100-fold, 250-fold; 500-fold; or 1,000-foldhigher than the concentration of the 5′-tailed primers.

The total primer concentration of the split-cycle amplification reactioncan be from about 1 nM to about 1 μM, from about 5 nM to about 900 nM,from about 10 nM to about 750 nM, from about 15 nM to about 600 nM, fromabout 20 nM to about 500 nM, from about 25 nM to about 400 nM, fromabout 50 nM to about 300 nM, from about 75 nM to about 250 nM, or fromabout 100 nM to about 200 nM. In some cases, the total primerconcentration in the split-cycle amplification reaction is, or is about,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 110, 120, 130,140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 350, 400, or 500nM.

Split-cycle amplification compositions and methods, such as thosedescribed herein, can increase the sensitivity of a nucleic acidamplification assay by, e.g., lowering variations due to mis-priming orpolymerase-mediated nucleotide mis-incorporation errors. For example, inan amplification reaction in which the 3′ hybridization regions of the5′-tailed primers hybridize to a target template sequence that is highlysimilar to a non-target sequence, early amplification cycles, in which5′-tailed primer extension predominates, can exhibit significantmis-priming of 5-tailed primer to non-target templates. Similarly,during polymerase-mediated nucleotide mis-incorporation hybridization ofa 5′-tailed primer to a target site can produce a non-target mutantprimer-extension product that is not, or not efficiently, amplified byadditional rounds of 5′-tailed primer-mediated amplification. Incontrast later amplification cycles, in which extension of flankingprimers predominates, can be highly resistant to mis-priming and/ormis-incorporation errors because the flanking primer binding site can besignificantly different from any other sequence in the sample.

By limiting the effect of mis-priming and/or mis-incorporation to asmall number of early amplification cycles, the assay can exhibitincreased sensitivity. Such errors due to mis-priming or polymerasenucleotide mis-incorporation be substantial during, e.g., variantanalysis, such as where the difference between target and non-target isa single nucleotide (e.g., SNP analysis). These mis-priming orpolymerase nucleotide mis-incorporation errors can also substantial in amulti-channel assays where two or more target templates differ by asmall number of nucleotides. For example, in an assay where mutant andtarget templates are separately detected, it can be beneficial to limitthe effects of mis-priming and/or mis-incorporation to a small number ofearly amplification cycles.

Accordingly, in some embodiments, the split-cycle reaction mixturesdescribed herein contain two sets of 5′-tailed forward and reverseprimer pairs and two sets of forward and reverse flanking primer pairs.For example, the split-cycle reaction mixtures described herein caninclude a wild-type specific 5′-tailed forward and reverse primer pairthat hybridizes to a target DNA template containing wild-type targetsequence and a mutation-specific 5′-tailed forward and reverse primerpair that hybridizes to a target DNA template containing a mutant targetsequence. Such reaction mixtures further contain two corresponding setsof flanking forward and reverse primer pairs.

In some cases, the difference between the wild-type target sequence andthe mutant target sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10nucleotides. Accordingly, in some cases, the hybridization regions ofthe wild-type and mutation-specific 5′-tailed forward primers differ by1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. Similarly, in some cases,the hybridization regions of the wild-type and mutation-specific5′-tailed reverse primers differ by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10nucleotides. In some cases, wild-type specific and mutation-specific5′-tailed forward primers and wild-type specific and mutation-specific5′-tailed reverse primers independently differ by, or by about, 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 nucleotides. In contrast, the 5′-tail regionsand flanking primers are generally selected to be substantiallydifferent from each other, such that flanking primers do notcross-hybridize to non-target tail regions under amplification reactionconditions.

In some cases, the mutation-specific 5′-tailed primers contain adiscriminatory nucleotide within the first 5 nucleotides of the 3′hybridization region, counting from the 3′ end, that discriminatebetween mutant and wild-type target sequence. For example, themutation-specific 5′-tailed primers can contain a discriminatorynucleotide that is the 3′-most nucleotide, the second-most 3′nucleotide, the third-most 3′ nucleotide, the fourth-most 3′ nucleotide,or the fifth-most 3′ nucleotide. In some cases, the mutation-specific5′-tailed primers contain multiple discriminatory nucleotides. In somecases, the discriminatory nucleotides are adjacent. In some cases, theforward and reverse 5′-tailed primers each contain a discriminatorynucleotide.

In some embodiments, a reaction mixture for performing a split-cyclenucleic acid amplification assay includes: a thermostable polymerase;any one or more of the foregoing target-specific forward and reverse5′-tailed primer pairs; and any one or more of the foregoingtarget-specific forward and reverse flanking primer pairs. In somecases, the target-specific forward and reverse 5′-tailed primer pairspecifically hybridizes to and amplifies a wild-type nucleic acid targetsequence. In some cases, the target-specific forward and reverse5′-tailed primer pair specifically hybridizes to and amplifies a mutantnucleic acid target sequence. In some cases, the reaction mixturecontains both a mutation-specific forward and reverse 5′-tailed primerpair and a wild-type specific forward and reverse 5′-tailed primer pair.In some cases, the reaction mixture contains one or more additionaltarget-specific forward and reverse 5′-tailed primer pairs thatspecifically hybridize to and amplify one or more additional targetnucleic acids. Reaction mixtures containing multiple forward and reverse5′-tailed primer pairs can contain a corresponding number of forward andreverse flanking primer pairs where each flanking primer pairspecifically amplifies amplicons generated by the corresponding5′-tailed primer pair.

In some embodiments, a reaction mixture for performing a split-cyclenucleic acid amplification assay includes: i) a target-specific forward5′-tailed primer and target-specific reverse 5′-tailed primer (i.e., atarget-specific 5′-tailed primer pair), where the target-specific5′-tailed primer pair hybridizes to and specifically amplifies targetDNA template molecules from a nucleic acid sample that contain thetarget sequence, if present, and where the primers of the targetspecific 5′-tailed primer pair include: a) a 3′ hybridization region ofat least 10 nucleotides in length and less than 30 nucleotides in lengththat specifically hybridizes to the mutant target sequence; and b) atarget-specific 5′ tail region of at least 10 nucleotides in length andno more than 30 nucleotides in length that does not hybridize to anynucleic acid fragments in the nucleic acid sample. In some cases, thereaction mixture further contains: ii) a pair of forward and reversetarget-specific flanking primers, wherein the target-specific flankingprimers hybridize to and specifically amplify amplicons containing the5′ tail regions of the target-specific 5′-tailed primer pair if present.

Split-cycle reaction mixtures can further contain amplification reagentsas known in the art. Such amplification reagents include, but are notlimited to, nucleotide triphosphates, divalent cation, buffer, salt,stabilizers, and other additives (e.g., betaine, DMSO, etc.). In somecases, the split-cycle reaction mixtures contain an intercalating dye,e.g., EvaGreen. Such intercalating dyes can be useful for detectingamplification in a reaction where single-channel detection is desired.For example, in a split-cycle amplification containing a single set of5′-tailed forward and reverse primers and a single set of correspondingflanking primers, an intercalating dye can discriminate between areaction mixture containing amplicons, indicating the presence of targetDNA template, and a reaction mixture that does not contain a substantialnumber of amplicons, indicating the absence of sufficient target DNAtemplate.

Alternatively, sequence specific probes can be present in thesplit-cycle reaction mixture. Sequence specific probes can be useful forsingle-channel detection as described above. Sequence specific probescan also be useful for multi-channel detection. For example, in ansplit-cycle assay in which two or more different amplicons are generatedusing two or more different sets of 5′-tailed primer pairs andcorresponding flanking primer pairs, sequence-specific probes candiscriminate between the two or more different amplicons. Exemplarysequence-specific probes include, but are not limited to hydrolysisprobes (e.g., TAQMAN), and Molecular Beacons. Alternatively, asdescribed above, the flanking primers can be detectably labeled sequencespecific probes. In some embodiments, reaction mixtures includingdetectably labeled flanking primers also include a sink oligonucleotidethat quenches the detectable label when hybridized to the flankingprimer. Disruption of the flanking primer:sink oligonucleotide hybridby, e.g., polymerase mediated hydrolysis, results in a detectable signalindicating the presence of the target nucleic acid.

b. Partitioned Split-Cycle Compositions

Split-cycle amplification can be performed on a partitioned nucleic acidsample in a plurality of mixture partitions. In some embodiments,accurate quantitation of target template molecules in the samplerequires that the plurality of mixture partitions produced bypartitioning of the nucleic acid sample is not saturated by the numberof target template molecules in the sample. As such, described hereinare split-cycle compositions containing a plurality of mixturepartitions, where the individual mixture partitions are generated bypartitioning the nucleic acid sample, and the plurality contains atleast about 1 mixture partition that does not contain a target templatemolecule (e.g., wild-type or mutant).

In some embodiments, the plurality contains about, or at least about,2-10 or 3-10 mixture partitions that do not contain a template molecule(e.g., wild-type or mutant). In some embodiments, the plurality containsabout 10; 20; 30; 40; 50; 60; 100; 1,000; 2,000; 3,000; 4,000; 5,000;10,000; 15,000, or more mixture partitions that do not contain atemplate molecule. In some embodiments about, or at least about, 0.001%,0.005%, 0.01%, 0.05%, 0.1%, 1%, 2%, 3%, 4%, 6%, 7%, 8%, 9%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%of the mixture partitions do not contain a template molecule. In someembodiments, from about 0.001% to about 50% of the mixture partitions donot contain a template molecule. In some embodiments, from about 0.005%to about 25% of the mixture partitions do not contain a templatemolecule. In some embodiments, from about 0.1% to about 15% of themixture partitions do not contain a template molecule. In someembodiments, from about 1% to about 10% of the mixture partitions do notcontain a template molecule. In some embodiments, from about 10% toabout 50% of the mixture partitions do not contain a template molecule.

In some embodiments, the target template molecules are present in theplurality of mixture partitions at a concentration of at least about0.000025 copies per partition. In some embodiments, the target templatemolecules are present in the plurality of mixture partitions at aconcentration of from about 0.000025 to about 50 copies per partition,from about 0.0001 to about 50 copies per partition, from about 0.0005 toabout 50 copies per partition, from about 0.001 to 40 copies perpartition, from about 0.005 to about 30 copies per partition, from about0.01 to about 20 copies per partition, or from about 0.00005 to about 20copies per partition. In some embodiments, the target template moleculesare present in the plurality of mixture partitions at a concentration offrom about 0.025 to about 30 copies per partition, from about 0.05 toabout 30 copies per partition, from about 0.01 to about 30 copies perpartition, from about 0.25 to about 30 copies per partition, from about0.025 to about 10 copies per partition, from about 0.05 to about 10copies per partition, from about 0.01 to about 10, or from about 0.25 toabout 10 copies per partition. In some cases, the target DNA template inthe mixture is at an average concentration of, or of about, 0.00025,0.0005, 0.00075, 0.001, 0.0025, 0.005, 0.0075, 0.01, 0.025, 0.05, 0.075,0.1, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 copies perpartition.

In some embodiments, the plurality of mixture partitions contains one ormore wild-type target template molecules. In some embodiments, theplurality of mixture partitions contains target template moleculeshaving a mutant target sequence. In some embodiments, the plurality ofmixture partitions contains at least one target template molecule havinga mutant target sequence and a plurality of wild-type target templatemolecules.

In some embodiments, at least one of the plurality of mixture partitionscontains both a wild-type target template molecule and a mutant targettemplate molecule. In some embodiments, about 0.0001%, or at least0.0001%, of the plurality of mixture partitions contains a wild-typetarget template molecule and a mutant target template molecule. In someembodiments, about 0.001%, or at least 0.001%, of the plurality ofmixture partitions contains a wild-type target template molecule and amutant target template molecule. In some embodiments, about 0.01%, or atleast 0.01%, of the plurality of mixture partitions contains a wild-typetarget template molecule and a mutant target template molecule. In someembodiments, about 0.1%, or at least 0.1%, of the plurality of mixturepartitions contains a wild-type target template molecule and a mutanttarget template molecule. In some embodiments, about 1%, or at least 1%,of the plurality of mixture partitions contains a wild-type targettemplate molecule and a mutant target template molecule. In someembodiments, about, or at least about, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45% of the plurality of mixturepartitions contains a wild-type target template molecule and a mutanttarget template molecule.

In some embodiments, the number of mixture partitions in the pluralityof mixture partitions is at least about 100. In some embodiments, thenumber of mixture partitions in the plurality of mixture partitions isfrom about 100 to about 10,000,000; from about 500 to about 10,000,000;from about 500 to about 200,000; from about 500 to about 100,000; fromabout 500 to about 75,000; from about 1,000 to about 50,000; or fromabout 5,000 to about 20,000. In some embodiments, the number of mixturepartitions in the plurality of mixture partitions is, or is about, 500;750; 1,000; 2,000; 3,000; 4,000; 5,000; 7,500; 5,000; 7,500; 10,000;15,000; 20,000; 30,000; 40,000; 50,000; 75,000; 100,000; 150,000;200,000; 500,000; 1,000,000; 5,000,000; or 10,000,000.

c. Tagged Amplicon Primer Extension (TAPE) Compositions

Described herein are compositions for performing tagged amplicon primerextension (TAPE) reactions. In one aspect, the composition is a set ofTAPE primers. TAPE primers are pairs of PCR nucleic acid amplificationprimers that contain 5′ tails, where the 5′ tails of the individualprimers of the pair are reverse complements of each other. In somecases, the TAPE primers are also split-cycle 5′-tailed primers.

TAPE forward and reverse primers can contain a 3′ hybridization regionthat is configured to specifically hybridize to a target sequence of atarget DNA template molecule and generate a first primer extensionproduct in a nucleic acid amplification reaction. In some cases, the 3′hybridization region is at least about 8 nucleotides in length. In somecases, the hybridization regions of the TAPE primers are 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45nucleotides in length. In some cases, the hybridization regions of theTAPE primers are from about 8 to about 45, about 9 to about 40, about 8to about 35, about 8 to about 30, about 9 to about 30, about 10 to about30, about 11 to about 25, about 12 to about 25, or about 15 to about 25,nucleotides in length.

TAPE forward and reverse primers can contain a 5′ tail region that isnot complementary to the target sequence of the target DNA templatemolecule. In some cases, the 5′ tail region is at least about 8nucleotides in length. In some cases, the 5′ tail regions of the TAPEprimers are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, or 45 nucleotides in length. In some cases, the 5′ tailregions of the TAPE primers are from about 8 to about 45, about 9 toabout 40, about 8 to about 35, about 8 to about 30, about 9 to about 30,about 10 to about 30, about 11 to about 25, about 12 to about 25, orabout 15 to about 25, nucleotides in length.

In some cases, the TAPE primers contain a discriminatory nucleotidewithin the first 5 nucleotides of the 3′ hybridization region, countingfrom the 3′ end, that discriminate between a mutant and wild-type targetsequence. For example, the discriminatory nucleotide can be the 3′-mostnucleotide, the second-most 3′ nucleotide, the third-most 3′ nucleotide,the fourth-most 3′ nucleotide, or the fifth-most 3′ nucleotide. In somecases, the TAPE primers contain multiple discriminatory nucleotides. Insome cases, the multiple discriminatory nucleotides are adjacent. Insome cases, the forward and reverse TAPE primers each contain adiscriminatory nucleotide.

In some embodiments, the 5′ tail regions of the forward and/or reverseTAPE primers contain a restriction endonuclease cleavage site. Therestriction endonuclease cleavage site can be useful to cleave ampliconconcatemers generated in the TAPE reaction. In some cases, both forwardand reverse TAPE primers contain a restriction endonuclease cleavagesite. In some cases, both forward and reverse TAPE primers contain thesame restriction endonuclease cleavage site. In some cases, the forwardand reverse TAPE primers contain different restriction endonucleasecleavage sites.

In some cases, the TAPE reaction mixture further contains a pair offorward and reverse flanking primers that specifically hybridizes to andamplifies amplicons containing the 5′ tail regions of the TAPE primers.

In some cases, the TAPE reaction contains a first forward and reverseTAPE primer pair that specifically hybridizes to and amplifies a firsttarget sequence and a second forward and reverse TAPE primer pair thatspecifically hybridizes to and amplifies a second target sequence.

In some cases, the first and second target sequences are a wild-type andcorresponding mutant target sequence respectively. Accordingly, thefirst TAPE primer pair can be a wild-type specific TAPE primer pairhaving a 3′ hybridization region that specifically hybridizes to andamplifies target template molecules having the wild-type targetsequence. In some cases, the forward and/or reverse primers of the firstTAPE primer pair can contain a discriminatory nucleotide in the 3′hybridization region that is complementary to the wild-type target DNAtemplate molecule but not the mutant target DNA template molecule.

Similarly, the second TAPE primer pair can be a mutation-specific TAPEprimer pair having a 3′ hybridization region that specificallyhybridizes to and amplifies target template molecules having a mutanttarget sequence. In some cases, the forward and/or reverse primers ofthe second TAPE primer pair can contain a discriminatory nucleotide inthe 3′ hybridization region that is complementary to the mutant targetDNA template molecule but not the wild-type target DNA templatemolecule.

In some cases, the 5′ tails of the first set of forward and reverse TAPEprimers are different from the 5′ tails of the second set of forward andreverse TAPE primers. In some cases, the TAPE reaction mixture furthercontains a first pair of forward and reverse flanking primers thatspecifically hybridizes to and amplifies amplicons containing the 5′tail regions of the first set of TAPE primers. In some cases, the TAPEreaction mixture further contains a second pair of forward and reverseflanking primers that specifically hybridizes to and amplifies ampliconscontaining the 5′ tail regions of the second set of TAPE primers.

TAPE reactions can contain any one or more of the foregoing nucleic acidamplification reaction compositions or mixtures described herein,including but not limited to, primers, probes, buffers, salts,polymerases, or the like, and combinations thereof. TAPE reactions canbe performed in a plurality of mixture partitions. As such, TAPEreaction compositions can include, but are not limited to, any one ormore of the foregoing partitioned split-cycle compositions describedherein, including but not limited to, those containing mutant andwild-type targets, those containing 1 or more partitions that do notcontain a target template molecule, those containing 1 or morepartitions that do contain a target template molecule, or thosecontaining 1 or more partitions that contain both a wild-type and targettemplate molecule, and combinations thereof.

III. Methods

a. Split-Cycle

Described herein are methods for quantitating an absolute number orfrequency of wild-type and mutant target nucleic acid fragments in anucleic acid sample. In some embodiments, the method includes forming aplurality of mixture partitions containing a fraction of the nucleicacid sample, one or more of the foregoing pairs of 5′-tailed primers,one or more of the foregoing pairs of flanking primers and athermostable polymerase. In some cases, the mixture partitions areemulsion droplets.

The method can include an incubation under thermal cycling conditionssuitable for amplification of target DNA template molecules, if present,by a polymerase chain reaction. In some cases, the thermal cyclingconditions include a first set of temperature cycles (e.g., a first setof denaturing, annealing, and extending) and a second set of temperaturecycles (e.g., a first set of denaturing, annealing, and extending),where the annealing step of the second set of temperature cycles is atleast 1° C. higher than the annealing step of the first set oftemperature cycles. In some cases, the annealing step of the second setof temperature cycles is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45° C. higher thanthe annealing step of the first set of temperature cycles. In somecases, the annealing step of the second set of temperature cycles isfrom about 1 to about 5° C., from about 1 to about 10° C., or from about1 to about 15° C. higher than the annealing step of the first set oftemperature cycles. In some cases, the annealing step of the second setof temperature cycles is from about 2 to about 5° C., from about 2 toabout 10° C., or from about 2 to about 15° C. higher than the annealingstep of the first set of temperature cycles. In some cases, theannealing step of the second set of temperature cycles is from about 5to about 10° C., from about 5 to about 15° C., or from about 5 to about20° C. higher than the annealing step of the first set of temperaturecycles.

In some cases, the thermal cycling conditions include a first set oftemperature cycles (e.g., a first set of denaturing, annealing, andextending) and a second set of temperature cycles (e.g., a first set ofdenaturing, annealing, and extending), where the annealing step of thesecond set of temperature cycles is at least 1° C. lower than theannealing step of the first set of temperature cycles. In some cases,the annealing step of the second set of temperature cycles is 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, or 45° C. lower than the annealing step of the first setof temperature cycles. In some cases, the annealing step of the secondset of temperature cycles is from about 1 to about 5° C., from about 1to about 10° C., or from about 1 to about 15° C. lower than theannealing step of the first set of temperature cycles. In some cases,the annealing step of the second set of temperature cycles is from about2 to about 5° C., from about 2 to about 10° C., or from about 2 to about15° C. lower than the annealing step of the first set of temperaturecycles. In some cases, the annealing step of the second set oftemperature cycles is from about 5 to about 10° C., from about 5 toabout 15° C., or from about 5 to about 20° C. lower than the annealingstep of the first set of temperature cycles.

In some embodiments, the method includes a step of detecting thepresence or absence of amplified target DNA template in the mixturepartitions. The detecting can thereby determine: i) the number ofsingle-positive wild-type mixture partitions containing amplified targetDNA template having a wild-type target sequence; ii) the number ofsingle-positive mutant mixture partitions containing amplified targetDNA template having a mutant target sequence; and iii) the number ofdouble-positive mixture partitions containing both wild-type and mutantamplified target DNA. In some cases, the numbers of single-positive anddouble-positive partitions are used to determine the absolute quantityor frequency of wild-type and mutant target nucleic acid fragments inthe nucleic acid sample. In some cases, the number of negativepartitions, or the total number of all partitions is also used tocalculate the absolute number or frequency of wild-type and mutanttarget nucleic acid fragments in the nucleic acid sample. The detectingcan be detecting fluorescence of an intercalating dye, detectingfluorescence of a fluorophore conjugated to a flanking primer (e.g.,with an AMP assay, see US 2015/0,148,250, herein incorporated byreference in the entirety for all purposes.), or detecting afluorescence signal generated by polymerase-mediated hydrolysis of anucleic acid hydrolysis probe.

In some embodiments, the first set of thermal cycling conditions is, orincludes at least, a single denaturing, annealing, and extension step.In some cases, the first set of thermal cycling conditions includes fromabout 1 about 20, from about 2 to about 15, or from about 2 to about 5cycles of denaturing, annealing, and extension. In some embodiments, thesecond set of thermal cycling conditions is, or includes at least, 5cycles of denaturing, annealing, and extension. In some cases, thesecond set of thermal cycling conditions includes from about 5 about 60,from about 10 to about 50, from about 15 to about 30, or from about 15to about 25 cycles of denaturing, annealing, and extension. In somecases, the flanking primers in the reaction mixture have an annealingtemperature at, or near (e.g., within about 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10° C.), an optimal extension temperature of the polymerase and theannealing and extension in the second set of temperature cycles areperformed simultaneously. In some cases, the 5′-tailed primers in thereaction mixture have an annealing temperature at, or near (e.g., withinabout 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10° C.), an optimal extensiontemperature of the polymerase and the annealing and extension in thefirst set of temperature cycles are performed simultaneously.

b. Tagged Amplicon Primer Extension (TAPE)

Described herein are methods for performing a TAPE reaction. In oneaspect, the method includes forming or providing any one of theforegoing reaction mixtures or plurality of mixture partitions. Thereaction mixtures or mixture partitions can include a thermostablepolymerase and a pair of forward and reverse TAPE primers having 5′tails that are reverse complements of each other. In some embodiments,the reaction mixture or plurality of mixture partitions, or a portionthereof, contain a target DNA template molecule.

The reaction mixture or plurality of mixture partitions can be subjectto conditions suitable to carry out hybridization (i.e., annealing) ofthe 3′ hybridizing regions of forward TAPE primers to target templatemolecules, if present. Hybridized TAPE primers can be extended with thepolymerase generating a first primer extension product. The first primerextension product can be hybridized to a reverse TAPE primer, which isthen extended with the polymerase, thereby generating a second primerextension product. The 3′ hybridizing regions of the forward TAPE primercan be hybridized to the second primer extension product and extended,thereby generating a third primer extension product. The second andthird primer extension products together form a first double-strandedamplicon having 3′ ends that are reverse complements of each other and5′ ends that are reverse complements of each other.

The method can further include denaturing the first double strandedamplicon and hybridizing the 3′ ends of the first double strandedamplicon to each other. The hybridized 3′ ends of the two strands of thefirst double stranded amplicon can be extended with the polymerase toform a second double stranded amplicon having 3′ ends that are reversecomplements of each other and 5′ ends that are reverse complements ofeach other. The second double-stranded amplicon can be larger than thefirst. The method can further include: denaturing the second doublestranded amplicon; hybridizing the 3′ ends of the two strands of thesecond double stranded amplicon; and extended the hybridized 3′ ends ofthe two strands of the second double-stranded amplicon with thepolymerase to generate a third double-stranded amplicon having 3′ endsthat are reverse complements of each other and 5′ ends that are reversecomplements of each other. The third double-stranded amplicon can belarger than the second.

The denaturing, annealing, and extending of double stranded ampliconscan be repeated a number of times, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, or 50, or more times. In some cases, the denaturing,annealing, and extending of double stranded amplicons is repeated fromabout 1 about 50, from about 2 to about 40, from about 5 to about 40,from about 10 to about 40, from about 15 to about 40, from about 20 toabout 40, from about 5 to about 30, from about 10 to about 30, fromabout 15 to about 30, from about 20 to about 30, from about 5 to about25, from about 10 to about 25, from about 15 to about 25, from about 5to about 20, from about 10 to about 20, or from about 15 to about 20cycles of denaturing, annealing, and extension. In some cases, thereverse complementary 3′ ends of the double-stranded amplicons in thereaction mixture have an annealing temperature at, or near (e.g., withinabout 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10° C.), an optimal extensiontemperature of the polymerase and the annealing and extension areperformed simultaneously.

In some embodiments, the large size of the amplicons generated in latecycles of the TAPE reaction results in precipitation. The precipitationcan be utilized to provide a readout of a presence or absence of atarget template molecule in a reaction mixture or mixture partition. Forexample, precipitation can be detected by optical detection of turbidityat an ultraviolet or visible wavelength. Additional or alternativemethods for detecting are described below. Alternatively,double-stranded amplicons can be cleaved to prevent or reduceprecipitation or to reduce amplicons to a uniform size.

Accordingly, in some cases, a cleavage site is included in the 5′ tailsof the TAPE primers. The cleavage site can be a restriction endonucleasesite, a uracil (e.g., cleavable by a uracil glycosylase enzyme), or achemically cleavable linkage. Generally, the cleavage site is selectedto minimize interference with template-directed DNA polymerase activityof the polymerase present in the reaction mixture or mixture partitions.In some cases, the cleavage site is a dialkoxysilane,3′-(S)-phosphorothioate, 5′-(S)-phosphorothioate,3′-(N)-phosphoramidate, 5′-(N)phosphoramidate linkage in the 5′ tailregion of one or both of the TAPE primers. In reaction mixtures ormixture partitions containing multiple pairs of TAPE primers, each TAPEprimer, or TAPE primer pair can contain a cleavage site in the 5′ tailregion independently selected from a dialkoxysilane,3′-(S)-phosphorothioate, 5′-(S)-phosphorothioate,3′-(N)-phosphoramidate, or 5′-(N)phosphoramidate linkage, a restrictionendonuclease cleavage site, and uracil. In some embodiments, some TAPEprimers or primer pairs in a reaction mixture or mixture partitioncontain a cleavage site in a 5′ tail and some do not.

TAPE primers containing cleavage sites in the 5′ tail regions generateamplicons containing a cleavage site. In some embodiments, the TAPEreaction method includes a step of cleaving the cleavage sites of theamplicons. The cleavage can be performed in the mixture partitions orreaction mixture. In some cases, the cleavage is performed by anextended incubation (e.g., 5, 10, 15, 30, 45, 60, or 90 minutes) at anoptimal cleavage enzyme temperature (e.g., 37° C.).

In some cases, the cleavage additionally or alternatively includesintroducing into the reaction mixture or mixture partitions a cleavagereagent (e.g., enzyme or chemical). For example, in some cases, anelectric field can be applied to an interface between a partition and afluid to disrupt the interface and allow at least a portion of the fluidto enter the partition. As another example, one or more reagents can bedirected to partitions in micro or nanoliter size wells via microfluidictechniques. Methods, compositions, and devices for injection of reagentsinto a partition can include, but are not limited to, those described inWO/2010/0151776.

TAPE amplification can be detected by detecting the presence ofamplicons or digested amplicons using a variety means known in the artto determine the presence or absence of a target template in thereaction mixture or plurality of mixture partitions. In some cases, theTAPE reaction amplifies both wild-type and mutant target templatemolecules, and the detecting is performed with a differentially labeledwild-type specific probe and a mutant-specific probe. The probes can benucleic acid hydrolysis probes, Molecular Beacons, Scorpion Probes, andthe like.

In some cases, the TAPE reaction amplifies both wild-type and mutanttarget template molecules because a single pair of forward and reverseTAPE primers can hybridize to both wild-type and mutant target templatemolecules and be extended to generate amplicons. In some cases, the TAPEreaction amplifies both wild-type and mutant target template moleculesusing two different pairs of forward and reverse TAPE primers, one pairspecifically amplifying wild-type target sequences and one pairspecifically amplifying mutant target sequence. In some cases, the TAPEreaction specifically amplifies a target template molecule and detectioncan be performed with a sequence specific probe or a non-specificdetection reagent such as an intercalating dye. In some cases, the TAPEreaction specifically amplifies a mutant target template molecule, e.g.,in the presence of wild-type target template molecules if also present,and detection can be performed with a sequence specific probe or anon-specific detection reagent such as an intercalating dye. In somecases, the sequence specific probe detects a region of the ampliconsthat is the same in the wild-type and mutant target templates.

In some cases, the TAPE reaction further includes flanking primers asdescribed above. In such TAPE reactions, the method can includesplit-cycle amplification methods and compositions as described above.For example TAPE amplification can be performed with a first set ofamplification conditions for hybridizing and extending TAPE primers andstrands of resulting double-stranded amplicons, and a second set ofamplification conditions for hybridizing and extending flanking primers.In some cases, the primer annealing temperature of the second set ofamplification conditions is higher than the annealing temperature of thefirst set of amplification conditions. In some cases, the primerannealing temperature of the second set of amplification conditions islower than the annealing temperature of the first set of amplificationconditions. In some cases, one or more flanking primers are detectablylabeled. In some cases, detection of amplification in such TAPEreactions can be performed using a, Amplicon Mediated Probe assay (AMPassay). Such assays are described further in US 2015/0,148,250, hereinincorporated by reference in the entirety for all purposes.

In some cases, a plurality of mixture partitions is provided orgenerated; and, a split-cycle reaction mixture, a TAPE amplificationreaction, or a combination thereof is performed to separately detectwild-type and mutant target DNA template molecules in the plurality ofmixture partitions, thereby detecting a number of mixture partitionsthat are positive for the presence of the mutant but not the wild-typetarget DNA template, a number of mixture partitions that are positivefor a presence of the wild-type but not the mutant target DNA template,a number of mixture partitions that are positive for the presence ofboth the mutant and wild-type target DNA template, and a number ofmixture partitions that are negative for the presence of mutant andwild-type target DNA template. The method can further includedetermining the frequency of the mutant sequence in the nucleic acidsample from the number of single-positive, double-positive, and negativemixture partitions.

c. General Partitioning Methods

Partitioning methods described in the section can be performed for TAPEreaction mixtures, split-cycle reaction mixtures, or reaction mixturesin which TAPE and split-cycle methods, or elements thereof, areperformed.

Partitions can include any of a number of types of partitions, includingsolid partitions (e.g., wells, reaction chambers, or tubes) and fluidpartitions (e.g., aqueous droplets within an oil phase). In someembodiments, the partitions are droplets. In some embodiments, thepartitions are micro channels. Methods and compositions for partitioninga sample are described, for example, in published patent applications WO2010/036352, US 2010/0173394, US 2011/0092373, and US 2011/0092376, theentire contents of each of which is incorporated by reference herein.

In some aspects, the number of partitions is chosen to ensure that aminority of, a substantial minority of, few, substantially no, or nopartitions contain multiple target template molecules, contain bothwild-type and mutant target template molecules, or both. In some aspect,the number of partitions is selected to ensure that a quantitativedigital amplification assay is not saturated (i.e., there remainspartitions that do not have any template molecules).

The number of partitions necessary to ensure adequate partitioning isdependent on a number of factors, including, but not limited to: (a) thenumber of template molecules in a nucleic acid sample; (b) the method ofpartitioning; and (e) the desired statistical significance. Partitioningof a nucleic acid sample containing template molecules such that few orno partitions contain multiple template molecules generally requirespartitioning under dilute conditions that generate a large number of“empty” partitions that do not contain any template molecules. Thus, insome embodiments, it is preferred to partition under conditions thatgenerate a significant number of partitions containing multiple targettemplate molecules. In general, the number of partitions is at leastabout 500; 1000; 10000; or 20,000; 30,000; 50,000; or more. In somecases, only about 3-10, or at least about 3-10, partitions do notcontain any target template molecules. In such cases, a significantnumber of partitions can contain multiple target template molecules. Insome cases, the multiple target template molecules in an individualpartition include both wild-type and mutant target template molecules.In some cases, the multiple target template molecules in an individualpartition are all wild-type. In some cases, the multiple target templatemolecules in an individual partition are all mutant.

In some embodiments, reagents such as salts (e.g., divalent cation),buffers, enzymes (e.g., cleavage enzymes), substrates, nucleotides,primers, etc. are mixed together (e.g., with a sample) prior topartitioning, and then the sample is partitioned. In some cases, thereagents include a polymerase and the sample is partitioned shortlyafter mixing reagents together so that substantially all, or themajority, of polymerase activity occurs after partitioning. In othercases, the reagents are mixed at a temperature in which the polymeraseproceeds slowly, or not at all, the sample is then partitioned, and thereaction temperature is adjusted to allow the polymerase reaction toproceed. For example, the reagents can be combined on ice, at less than5° C., or at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 20-25, 25-30, or 30-35° C. or more. In general, one ofskill in the art will know how to select a temperature at which one ormore polymerase enzymes are not active. In some cases, a combination oftemperature and time are utilized to avoid substantial polymeraseactivity prior to partitioning.

In some cases, reagents can be mixed using one or more hot startpolymerases, such as a hot start DNA-dependent DNA polymerase. Thus,buffers, salts, nucleotides, labels, primers, enzymes, etc. can be mixedand then partitioned. Subsequently, the polymerization reaction,including multiple rounds of polymerization and/or amplification, can beinitiated by heating the partition mixtures to activate the one or morehot-start polymerases.

Additionally, reagents can be mixed together without one or morereagents necessary to initiate an enzymatic reaction (e.g.,polymerization and/or amplification). The mixture can then bepartitioned into a set of first partition mixtures and then the one ormore essential reagents can be provided by fusing the set of firstpartition mixtures with a set of second partition mixtures that providethe essential reagent. Alternatively, the essential reagent can be addedto the first partition mixtures without forming second partitionmixtures. For example, the essential reagent can diffuse into the set offirst partition mixture water-in-oil droplets. As another example, themissing reagent can be directed to a set of micro channels which containthe set of first partition mixtures.

In some embodiments, reagents can be mixed together to form a reactionmixture, and partitioned. Subsequently, one or more additional reagentscan be added to the partitions. For example, one or more reagents can beinjected into the partitions. In some cases, an electric field can beapplied to an interface between a partition and a fluid to disrupt theinterface and allow at least a portion of the fluid to enter thepartition. As another example, one or more reagents can be directed topartitions in micro or nanoliter size wells via microfluidic techniques.Methods, compositions, and devices for injection of reagents into apartition can include, but are not limited to, those described inWO/2010/0151776. Reagents that can be added by fusing partitions,injection, microfluidics or other means include but are not limited toamplification reagents, detection reagents, or combinations thereof. Forexample, DNA-dependent DNA polymerase (and, optionally, one or moreprimers) can be added into a partition to amplify a target templatenucleic acid in the partition.

In some embodiments, a droplet comprises an emulsion composition, i.e.,a mixture of immiscible fluids (e.g., water and oil). In someembodiments, a droplet is an aqueous droplet that is surrounded by animmiscible carrier fluid (e.g., oil). In some embodiments, a droplet isan oil droplet that is surrounded by an immiscible carrier fluid (e.g.,an aqueous solution). In some embodiments, the droplets described hereinare relatively stable and have minimal coalescence between two or moredroplets. In some embodiments, less than 0.0001%, 0.0005%, 0.001%,0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or10% of droplets generated from a sample coalesce with other droplets.The emulsions can also have limited flocculation, a process by which thedispersed phase comes out of suspension in flakes. In some embodiments,the droplet is formed by flowing an oil phase through an aqueous samplecomprising one or more of the compositions described herein.

The oil phase can comprise a fluorinated base oil which can additionallybe stabilized by combination with a fluorinated surfactant such as aperfluorinated polyether. In some embodiments, the base oil comprisesone or more of a HFE 7500, FC-40, FC-43, FC-70, or another commonfluorinated oil. In some embodiments, the oil phase comprises an anionicfluorosurfactant. In some embodiments, the anionic fluorosurfactant isAmmonium Krytox (Krytox-AS), the ammonium salt of Krytox FSH, or amorpholino derivative of Krytox FSH. Krytox-AS can be present at aconcentration of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%,0.9%, 1.0%, 2.0%, 3.0%, or 4.0% (w/w). In some embodiments, theconcentration of Krytox-AS is about 1.8%. In some embodiments, theconcentration of Krytox-AS is about 1.62%. Morpholino derivative ofKrytox FSH can be present at a concentration of about 0.1%, 0.2%, 0.3%,0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, or 4.0% (w/w). Insome embodiments, the concentration of morpholino derivative of KrytoxFSH is about 1.8%. In some embodiments, the concentration of morpholinoderivative of Krytox FSH is about 1.62%.

In some embodiments, the oil phase further comprises an additive fortuning the oil properties, such as vapor pressure, viscosity, or surfacetension. Non-limiting examples include perfluorooctanol and1H,1H,2H,2H-Perfluorodecanol. In some embodiments,1H,1H,2H,2H-Perfluorodecanol is added to a concentration of about 0.05%,0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%,0.8%, 0.9%, 1.0%, 1.25%, 1.50%, 1.75%, 2.0%, 2.25%, 2.5%, 2.75%, or 3.0%(w/w). In some embodiments, 1H,1H,2H,2H-Perfluorodecanol is added to aconcentration of about 0.18% (w/w).

In some embodiments, the emulsion is formulated to produce highlymonodisperse droplets having a liquid-like interfacial film that can beconverted by heating into microcapsules having a solid-like interfacialfilm; such microcapsules can behave as bioreactors able to retain theircontents through an incubation period. The conversion to microcapsuleform can occur upon heating. For example, such conversion can occur at atemperature of greater than about 40°, 50°, 60°, 70°, 80°, 90°, or 95°C. During the heating process, a fluid or mineral oil overlay can beused to prevent evaporation. Excess continuous phase oil can be removedprior to heating, or left in place. The microcapsules can be resistantto coalescence and/or flocculation across a wide range of thermal andmechanical processing.

Following conversion of droplets into microcapsules, the microcapsulescan be stored at about 70°, —20°, 0°, 3°, 4°, 5°, 6°, 7°, 8°, 9°, 10°,15°, 20°, 25°, 30°, 35°, or 40° C. In some embodiments, these capsulesare useful for storage or transport of partition mixtures. For example,samples can be collected at one location, partitioned into dropletscontaining enzymes, buffers, and/or primers or other probes, optionallyone or more polymerization reactions can be performed, the partitionscan then be heated to perform microencapsulation, and the microcapsulescan be stored or transported for further analysis.

The microcapsule partitions can resist coalescence, particularly at hightemperatures. Accordingly, the capsules can be incubated at a very highdensity (e.g., number of partitions per unit volume). In someembodiments, greater than 100,000, 500,000, 1,000,000, 1,500,000,2,000,000, 2,500,000, 5,000,000, or 10,000,000 partitions can beincubated per mL. In some embodiments, the incubations occur in a singlewell, e.g., a well of a microtiter plate, without inter-mixing betweenpartitions. The microcapsules can also contain other componentsnecessary for a reaction to occur during the incubation.

In some embodiments, a sample containing one or more of the compositionsdescribed herein is partitioned into at least 500 partitions, at least1000 partitions, at least 2000 partitions, at least 3000 partitions, atleast 4000 partitions, at least 5000 partitions, at least 6000partitions, at least 7000 partitions, at least 8000 partitions, at least10,000 partitions, at least 15,000 partitions, at least 20,000partitions, at least 30,000 partitions, at least 40,000 partitions, atleast 50,000 partitions, at least 60,000 partitions, at least 70,000partitions, at least 80,000 partitions, at least 90,000 partitions, atleast 100,000 partitions, at least 200,000 partitions, at least 300,000partitions, at least 400,000 partitions, at least 500,000 partitions, atleast 600,000 partitions, at least 700,000 partitions, at least 800,000partitions, at least 900,000 partitions, at least 1,000,000 partitions,at least 2,000,000 partitions, at least 3,000,000 partitions, at least4,000,000 partitions, at least 5,000,000 partitions, at least 10,000,000partitions, at least 20,000,000 partitions, at least 30,000,000partitions, at least 40,000,000 partitions, at least 50,000,000partitions, at least 60,000,000 partitions, at least 70,000,000partitions, at least 80,000,000 partitions, at least 90,000,000partitions, at least 100,000,000 partitions, at least 150,000,000partitions, or at least 200,000,000 partitions.

In some embodiments, a sample containing one or more of the compositionsdescribed herein is partitioned into a sufficient number of partitionssuch that all, substantially all, or at least a majority of partitionshave no more than 1 mutant target template molecule.

In some embodiments, emulsion droplet partitions that are generated aresubstantially uniform in shape and/or size. For example, in someembodiments, the droplets are substantially uniform in average diameter.In some embodiments, the droplets that are generated have an averagediameter of about 0.001 microns, about 0.005 microns, about 0.01microns, about 0.05 microns, about 0.1 microns, about 0.5 microns, about1 microns, about 5 microns, about 10 microns, about 20 microns, about 30microns, about 40 microns, about 50 microns, about 60 microns, about 70microns, about 80 microns, about 90 microns, about 100 microns, about150 microns, about 200 microns, about 300 microns, about 400 microns,about 500 microns, about 600 microns, about 700 microns, about 800microns, about 900 microns, or about 1000 microns.

In some embodiments, the droplets that are generated have an averagediameter of less than about 1000 microns, less than about 900 microns,less than about 800 microns, less than about 700 microns, less thanabout 600 microns, less than about 500 microns, less than about 400microns, less than about 300 microns, less than about 200 microns, lessthan about 100 microns, less than about 50 microns, or less than about25 microns. In some embodiments, the droplets that are generated arenon-uniform in shape and/or size.

In some embodiments, the mixture partitions (e.g., emulsion dropletpartitions) that are generated are substantially uniform in volume. Forexample, the standard deviation of mixture partition volume (e.g.,droplet volume) can be less than about 1 picoliter, 5 picoliters, 10picoliters, 100 picoliters, 1 nL, or less than about 10 nL. In somecases, the standard deviation of mixture partition volume (e.g., dropletvolume) can be less than about 10-25% of the average droplet volume. Insome embodiments, the mixture partitions (e.g., droplets) that aregenerated have an average volume of about 0.001 nL, about 0.005 nL,about 0.01 nL, about 0.02 nL, about 0.03 nL, about 0.04 nL, about 0.05nL, about 0.06 nL, about 0.07 nL, about 0.08 nL, about 0.09 nL, about0.1 nL, about 0.2 nL, about 0.3 nL, about 0.4 nL, about 0.5 nL, about0.6 nL, about 0.7 nL, about 0.8 nL, about 0.9 nL, about 1 nL, about 1.5nL, about 2 nL, about 2.5 nL, about 3 nL, about 3.5 nL, about 4 nL,about 4.5 nL, about 5 nL, about 5.5 nL, about 6 nL, about 6.5 nL, about7 nL, about 7.5 nL, about 8 nL, about 8.5 nL, about 9 nL, about 9.5 nL,about 10 nL, about 11 nL, about 12 nL, about 13 nL, about 14 nL, about15 nL, about 16 nL, about 17 nL, about 18 nL, about 19 nL, about 20 nL,about 25 nL, about 30 nL, about 35 nL, about 40 nL, about 45 nL, orabout 50 nL.

Examples

A PIK3CA mutation assay was performed on a sample to detect raremutations. FIG. 1 illustrates exemplary primer design for wild-type andmutation-specific primers for performing split-cycle up amplification.FIG. 2 illustrates exemplary primer design for wild-type anmutation-specific primers for performing split-cycle down amplification.FIGS. 3a-b illustrate exemplary primer designs and amplificationreaction schemes for performing a TAPE assay. FIG. 4 illustrates typicalresults expected of a rare mutation detection assay using conventionaldroplet digital amplification methods (Left) and a split-cycle, TAPE, orsplit-cycle and TAPE assay (Right) as described herein. In theconventional assay, double-negative droplets (black), single-positivemutant droplets (blue), double-positive droplets (mutant an wild-typetemplate present in the droplet) (brown), and single-positive wild-typedroplets (green) smear into each other and can be difficult to reliablydistinguish at the regions bordering between two different categories ofdroplets. In contrast, for the split-cycle and/or TAPE assay,double-negative droplets (black), single-positive mutant droplets(blue), double-positive droplets (mutant an wild-type template presentin the droplet) (brown), and single-positive wild-type droplets (green)are orthogonally situated to increase separation between droplets anincrease the number of droplets that can be assigned to a specificcategory.

FIG. 5 illustrates results of a PIK3CA detection assay that wasperformed using conventional droplet digital amplification methods(Left) and a split-cycle assay (Right). The results illustrated in FIG.5 were from a non-optimized thermal cycling protocol an reactionmixture. Further improvement can be provided by optimizing the first setof annealing and extension temperatures an second set of annealing anextension temperatures, as well as 5′-tailed an flanking primerconcentrations using routine optimization methods known in the art andin view of the methods and compositions described herein. In theconventional assay, double-negative droplets (black), single-positivemutant droplets (blue), double-positive droplets (mutant an wild-typetemplate present in the droplet) (brown), and single-positive wild-typedroplets (green) smeared into each other and can be difficult toreliably distinguish at the regions bordering between two differentcategories of droplets. In contrast, for the split-cycle assay,double-negative droplets (black), single-positive mutant droplets(blue), double-positive droplets (mutant an wild-type template presentin the droplet) (brown), and single-positive wild-type droplets (green)were orthogonally situated to increase separation between droplets anincrease the number of droplets that can be assigned to a specificcategory, thereby improving the accuracy and sensitivity of the assay.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications may be practiced within the scope of theappended claims. All patents, patent applications, and otherpublications, including GenBank Accession Numbers, cited in thisapplication are incorporated by reference in the entirety for allpurposes.

1. A plurality of mixture partitions, the individual mixture partitionscomprising: i) a mutation-specific 5′-tailed primer pair, wherein themutation-specific 5′-tailed primer pair hybridizes to and specificallyamplifies target DNA template molecules from a nucleic acid sample thatcomprise a mutant target sequence, if present, wherein the primers ofthe mutation specific 5′-tailed primer pair comprise: a) a 3′hybridization region of at least 10 nucleotides in length and less than30 nucleotides in length that specifically hybridizes to the mutanttarget sequence; and b) a mutation-specific 5′ tail region of at least10 nucleotides in length and no more than 30 nucleotides in length thatdoes not hybridize to any nucleic acid fragments in the nucleic acidsample; ii) a wild-type specific 5′-tailed primer pair, wherein thewild-type specific 5′-tailed primer pair hybridizes to and specificallyamplifies target DNA template molecules comprising a wild-type targetsequence, if present, wherein the primers of the wild-type specific5′-tailed primer pair comprise: a) a 3′ hybridization region of at least10 nucleotides in length and less than 30 nucleotides in length thatspecifically hybridizes to the wild-type target sequence; and b) awild-type specific 5′ tail region of at least 10 nucleotides in lengthand no more than 30 nucleotides that does not hybridize to any nucleicacid fragments in the nucleic acid sample, wherein the wild-typespecific 5′ tail region is a different sequence than themutation-specific 5′ tail region; and iii) a mutation specific flankingprimer pair, wherein the mutation specific flanking primer pairhybridizes to and specifically amplifies amplicons comprising the 5′tail regions of the mutation specific 5′-tailed primer pair, if present;iv) a wild-type specific flanking primer pair, wherein the wild-typespecific flanking primer pair hybridizes to and specifically amplifiesamplicons comprising the 5′ tail regions of the wild-type specific5′-tailed primer pair, if present; and v) a thermostable polymerase,wherein at least about 1-10 of the mixture partitions of the pluralityof mixture partitions contains a target DNA template molecule thatcomprises a wild-type or mutant target sequence; and at least about 3-10of the mixture partitions of the plurality of mixture partitions do notcontain the target DNA template molecule.
 2. The plurality of mixturepartitions of claim 1, wherein the mixture partitions comprise emulsiondroplets. 3-4. (canceled)
 5. The plurality of mixture partitions ofclaim 1, wherein at least about 10% and no more than about 90% of theplurality of mixture partitions comprise multiple target DNA templatemolecules.
 6. (canceled)
 7. The plurality of mixture partitions of claim1 wherein the target DNA template in the mixture partitions is at anaverage concentration of about 1-5 copies per partition.
 8. Theplurality of mixture partitions of claim 1, wherein at least one of thetarget DNA templates in the mixture partitions comprises a mutant targetsequence. 9-10. (canceled)
 11. The plurality of mixture partitions ofclaim 1, wherein the plurality comprises from about 500 to about10,000,000 mixture partitions.
 12. (canceled)
 13. The plurality ofmixture partitions of claim 1, wherein the 3′ hybridization regions ofthe wild-type and mutant 5′-tailed primer pairs comprise annealingtemperatures to the wild-type and mutant target DNA template moleculesrespectively that are at least about 5° C. less than the annealingtemperatures of the mutation specific and wild-type specific flankingprimers. 14-19. (canceled)
 20. The plurality of mixture partitions ofclaim 1, wherein the 3′ hybridization regions of the wild-type andmutant 5′-tailed primer pairs comprise annealing temperatures to thewild-type and mutant target DNA template molecules respectively that areat least about 5° C. more than the annealing temperatures of themutation specific and wild-type specific flanking primers. 21-26.(canceled)
 27. A method for quantitating a frequency of wild-type andmutant target nucleic acid fragments in a nucleic acid sample, themethod comprising: (a) forming a plurality of mixture partitions of anyone of the preceding claims; (b) incubating the mixture partitions underthermal cycling conditions suitable for amplification of the target DNAtemplate molecules by a polymerase chain reaction, wherein the thermalcycling conditions comprise a first set of temperature cycles and asecond set of temperature cycles, wherein the second set of temperaturecycles comprises an annealing temperature that is at least 5° C. higheror lower than an annealing temperature of the first set of temperaturecycles; (c) detecting the presence or absence of amplified target DNAtemplate in the mixture partitions, wherein the detecting comprisesdetermining: i) a number of wild-type mixture partitions comprisingamplified target DNA template consisting of wild-type target sequence;ii) a number of mutant mixture partitions comprising amplified targetDNA template consisting of mutant target sequence; and iii) a number ofdouble-positive mixture partitions comprising amplified target DNAcomprising mutant and wild-type target sequence; and (d) determiningfrom the number of wild-type, mutant, and double-positive mixturepartitions the frequency of wild-type and mutant target nucleic acidfragments in the nucleic acid sample. 28-31. (canceled)
 32. A reactionmixture for performing a tagged amplicon primer extension (TAPE) nucleicacid amplification reaction, the mixture comprising: i) a target DNAtemplate molecule from a nucleic acid sample, wherein the target DNAtemplate molecule comprises a target sequence; ii) a forward primercomprising: a) a 3′ hybridization region of at least 10 nucleotides inlength and no more than 30 nucleotides in length that is configured tospecifically hybridize to the target sequence of the target DNA templatemolecule and generate a first primer extension product in the nucleicacid amplification reaction; and b) a 5′ tail region of at least 10nucleotides in length that is not complementary to the target sequenceof the target DNA template molecule; iii) a reverse primer comprising:a) a 3′ hybridization region of at least 10 nucleotides in length and nomore than 30 nucleotides in length that is configured to specificallyhybridize to the first primer extension product and generate a secondprimer extension product in in the nucleic acid amplification reaction;and b) a 5′ tail region of at least 10 nucleotides in length that is notcomplementary to the target sequence of the target DNA templatemolecule, wherein the 5′ tail region of the reverse primer is a reversecomplement of the 5′ tail region of the forward primer; and iv) athermostable polymerase.
 33. The reaction mixture of claim 32, whereinthe thermostable polymerase comprises 3′ to 5′ exonuclease activity. 34.The reaction mixture of claim 32, wherein the 3′ hybridization region ofthe forward or reverse primer comprises a discriminatory nucleotide thatis complementary to a mutant target DNA template molecule comprising amutant target sequence but not complementary to a wild-type target DNAtemplate molecule comprising a wild-type target sequence.
 35. Thereaction mixture of claim 32, wherein the 3′ hybridization region of theforward and reverse primers comprise a discriminatory nucleotide that iscomplementary to the mutant target DNA template molecule but not thewild-type target DNA template molecule.
 36. (canceled)
 37. The reactionmixture of claim 32, wherein the 5′ tail region of the forward orreverse primers comprises a restriction endonuclease cleavage site.38-39. (canceled)
 40. The reaction mixture of claim 32, wherein thereaction mixture comprises: a mutation-specific forward and reverseprimer pair, each primer of the pair comprising a 3′ hybridizationregion comprising a discriminatory nucleotide that is complementary to amutant target DNA template molecule but not a wild-type target DNAtemplate molecule; and a wild-type specific forward and reverse primerpair, each primer of the pair comprising a 3′ hybridization regioncomprising a discriminatory nucleotide that is complementary to thewild-type target DNA template molecule but not the mutant target DNAtemplate molecule.
 41. The reaction mixture of claim 40, wherein the 5′tail regions of the mutation-specific pair of forward and reverseprimers are different from the 5′ tail regions of the wild-type specificpair of forward and reverse primers, and wherein the reaction mixturefurther comprises: a mutation-specific flanking primer pair, wherein themutation-specific flanking primer pair hybridizes to and specificallyamplifies amplicons comprising the 5′ tail regions of themutation-specific forward and reverse primer pair; and a wild-typespecific flanking primer pair, wherein the wild-type specific flankingprimer pair hybridizes to and specifically amplifies ampliconscomprising the 5′ tail regions of the wild-type specific forward andreverse primer pair.
 42. A plurality of mixture partitions, theindividual mixture partitions comprising the reaction mixture of claim32. 43-45. (canceled)
 46. A method for performing a tagged ampliconprimer extension (TAPE) nucleic acid amplification reaction, the methodcomprising: i) forming a reaction mixture of claim 32; ii) hybridizing aforward primer to a target sequence of a target DNA template molecule;iii) extending the hybridized forward primer with a polymerase, therebygenerating a first primer extension product; iv) hybridizing a reverseprimer to the first primer extension product; v) extending thehybridized reverse primer with the polymerase, thereby generating asecond primer extension product; vi) hybridizing the forward primer tothe second primer extension product; v) extending the forward primerhybridized to the second primer extension product with the polymerase,thereby generating a third primer extension product, wherein the secondand third primer extension products form a first double-strandedamplicon, wherein the first double-stranded amplicon comprises twocomplementary strands having 3′ and 5′ ends, wherein the 3′ ends arereverse complements of each other, and the 5′ ends are reversecomplements of each other. 47-54. (canceled)
 55. A method for performinga tagged amplicon primer extension (TAPE) nucleic acid amplificationreaction, the method comprising: i) forming a reaction mixture of claim41; ii) hybridizing: a) a mutant-specific forward primer to a mutanttarget sequence of a target DNA template molecule, if present; and b) awild-type specific forward primer to a wild-type target sequence of atarget DNA template molecule; iii) extending the hybridized forwardprimer(s) with a polymerase, thereby generating a mutant first primerextension product if the mutant target sequence is present and awild-type first primer extension product; iv) hybridizing: a) amutant-specific reverse primer to the mutant first primer extensionproduct, if present; and b) a wild-type specific reverse primer to thewild-type first primer extension product; v) extending the hybridizedreverse primer(s) with the polymerase, thereby generating a mutantsecond primer extension product if the mutant target sequence ispresent, and a wild-type second primer extension product; vi)hybridizing the forward primer(s) to the second primer extensionproduct(s), if present; v) extending the forward primer(s) hybridized tothe second primer extension product(s) with the polymerase, therebygenerating a mutant third primer extension product, if the mutant targetsequence is present and a wild-type third primer extension product,wherein: the second and third mutant primer extension products form amutant double-stranded amplicon, if the mutant target sequence ispresent, wherein the mutant double-stranded amplicon comprises twocomplementary strands having 3′ and 5′ ends, wherein the 3′ ends arereverse complements of each other, and the 5′ ends are reversecomplements of each other; and the second and third wild-type primerextension products form a wild-type double-stranded amplicon, whereinthe wild-type double-stranded amplicon comprises two complementarystrands having 3′ and 5′ ends, wherein the 3′ ends of the firstwild-type double-stranded amplicon are reverse complements of each otherand the 5′ ends of the first wild-type double-stranded amplicon arereverse complements of each other. 56-58. (canceled)
 59. A method forquantitative rare mutation detection, the method comprising: i)providing a plurality of mixture partitions of claim 42; ii) performinga split-cycle assay, TAPE assay, or combination thereof to separatelydetect wild-type and mutant target DNA template molecules in theplurality of mixture partitions, thereby detecting a number of mixturepartitions that are positive for a presence of the mutant but not thewild-type target DNA template, a number of mixture partitions that arepositive for a presence of the wild-type but not the mutant target DNAtemplate, a number of mixture partitions that are positive for thepresence of both the mutant and wild-type target DNA template, and anumber of mixture partitions that are negative for the presence ofmutant and wild-type target DNA template; and iii) determining thefrequency of the mutant sequence in the nucleic acid sample from thenumber of single-positive, double-positive, and negative mixturepartitions. 60-66. (canceled)